Limits...
Differential expression of immunogenic proteins on virulent Mycobacterium tuberculosis clinical isolates.

Schierloh P, Klepp L, Vazquez C, Rocha RV, Blanco FC, Balboa L, López B, Ritacco V, Bigi F, Sasiain Mdel C - Biomed Res Int (2014)

Bottom Line: Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response.We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins.Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB.

View Article: PubMed Central - PubMed

Affiliation: IMEX-CONICET, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 CABA, Argentina.

ABSTRACT
Molecular epidemiology has revealed that Mycobacterium tuberculosis (Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was also correlated with anti-Mtb-cell surface IgM, but not with anti-ManLAM, anti-PPD, or anti-Mtb-surface IgG antibodies. Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB.

Show MeSH

Related in: MedlinePlus

Antibodies against Rv2241, Rv0009, Rv0407, and Rv2624c recombinant proteins in drug-sensitive and MDR-TB patients' sera. ELISA plates were coated with recombinant proteins, loaded with 1/500 serum dilutions, and reveled with HRP conjugated anti-human polyvalent IgM/A/G. (a) 1D scatter plots indicating serological results of No-TB (No TB, n = 31), drug-sensitive TB (S TB, n = 19), and multi-drug-resistant TB (MDR TB, n = 18) patients' sera. Threshold, defined by maximum likelihood ratio (ROC analysis), was shaded in gray. Seropositive samples for each protein were color highlighted (Rv2241 = red, Rv0009 = purple, Rv0407 = blue, and Rv2624c = green). (b) ROC analysis indicating area under curve ± 95% CI. (c) Correlation analysis between signals (mean ± SEM) of positive samples versus immune-informatics predicted epitope density (median ± range) for each recombinant protein.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109345&req=5

fig3: Antibodies against Rv2241, Rv0009, Rv0407, and Rv2624c recombinant proteins in drug-sensitive and MDR-TB patients' sera. ELISA plates were coated with recombinant proteins, loaded with 1/500 serum dilutions, and reveled with HRP conjugated anti-human polyvalent IgM/A/G. (a) 1D scatter plots indicating serological results of No-TB (No TB, n = 31), drug-sensitive TB (S TB, n = 19), and multi-drug-resistant TB (MDR TB, n = 18) patients' sera. Threshold, defined by maximum likelihood ratio (ROC analysis), was shaded in gray. Seropositive samples for each protein were color highlighted (Rv2241 = red, Rv0009 = purple, Rv0407 = blue, and Rv2624c = green). (b) ROC analysis indicating area under curve ± 95% CI. (c) Correlation analysis between signals (mean ± SEM) of positive samples versus immune-informatics predicted epitope density (median ± range) for each recombinant protein.

Mentions: The following antigens (Ags) were employed: mannosylated lipoarabinomannan (ManLAM) from H37Rv strain (kindly provided by J. Belisle, Colorado State University, Fort Collins, CO, USA); protein purified derivative (PPD) (Statens Serum Institut, Copenhagen, Denmark). Homemade indirect enzyme-linked immunosorbent assay (ELISA) was used for detection of serum Abs employing goat anti-human polyvalent (anti-IgA/IgM/IgG) horseradish peroxidase- (HRP-) conjugated secondary Ab as detection reagent (Sigma-Aldrich Co. St. Louis, MO, USA). Briefly, 96-well high binding microplates (Corning, USA) were coated overnight (ON) at 4°C with 100 μL of the following Ags: PPD (1 μg/mL, in CO3 buffer pH = 9), ManLAM (0.5 μg/mL, in PO2 buffer pH = 8), and recombinant proteins (1 μg/mL, in PBS pH = 7.4). Optimal Ag concentration and coating buffer was experimentally determined (37, and data not shown). After properly washing with PBS 0.05% (v/v) tween-20 (Sigma) and blocking with PBS 2% (v/v) human serum albumin (HSA) (NatoCor, Córdoba, Argentina) plates were seeded and ON incubated at 4°C with serum dilutions: 1/1000 (PPD), 1/800 (ManLAM), and 1/500 (recombinant proteins). Serum dilutions were established in preliminary assays. Each sample was tested in duplicate. In assays with recombinant proteins (Figure 3), unspecific Abs were depleted from sera by adsorption on plates coated with glutathione-S-transferase (GST) (1 μg/mL, in PBS pH = 7.4) for 1 h at 37°C before serum-Ag incubation step. To deplete Abs directed to surface exposed Mtb-Ags from sera samples (Figure 5(b)), 150 μL of 1/500 diluted sera was also preadsorbed with 106  γ-irradiated H12435 or LAM10406 clinical isolates for 1 h at 37°C before serum-Ag incubation step. Finally, secondary reagent (100 μL, 1/3000 dilution) was added for 30 min and, after extensive washing, HRP activity was measured using tetramethylbenzidine (TMB) substrate (Sigma). Absorbance was measured at 450 nm (subtracting 570 nm background signal) using an UVM340 plate reader with DigiRead software (Asys Hitech, UK).


Differential expression of immunogenic proteins on virulent Mycobacterium tuberculosis clinical isolates.

Schierloh P, Klepp L, Vazquez C, Rocha RV, Blanco FC, Balboa L, López B, Ritacco V, Bigi F, Sasiain Mdel C - Biomed Res Int (2014)

Antibodies against Rv2241, Rv0009, Rv0407, and Rv2624c recombinant proteins in drug-sensitive and MDR-TB patients' sera. ELISA plates were coated with recombinant proteins, loaded with 1/500 serum dilutions, and reveled with HRP conjugated anti-human polyvalent IgM/A/G. (a) 1D scatter plots indicating serological results of No-TB (No TB, n = 31), drug-sensitive TB (S TB, n = 19), and multi-drug-resistant TB (MDR TB, n = 18) patients' sera. Threshold, defined by maximum likelihood ratio (ROC analysis), was shaded in gray. Seropositive samples for each protein were color highlighted (Rv2241 = red, Rv0009 = purple, Rv0407 = blue, and Rv2624c = green). (b) ROC analysis indicating area under curve ± 95% CI. (c) Correlation analysis between signals (mean ± SEM) of positive samples versus immune-informatics predicted epitope density (median ± range) for each recombinant protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109345&req=5

fig3: Antibodies against Rv2241, Rv0009, Rv0407, and Rv2624c recombinant proteins in drug-sensitive and MDR-TB patients' sera. ELISA plates were coated with recombinant proteins, loaded with 1/500 serum dilutions, and reveled with HRP conjugated anti-human polyvalent IgM/A/G. (a) 1D scatter plots indicating serological results of No-TB (No TB, n = 31), drug-sensitive TB (S TB, n = 19), and multi-drug-resistant TB (MDR TB, n = 18) patients' sera. Threshold, defined by maximum likelihood ratio (ROC analysis), was shaded in gray. Seropositive samples for each protein were color highlighted (Rv2241 = red, Rv0009 = purple, Rv0407 = blue, and Rv2624c = green). (b) ROC analysis indicating area under curve ± 95% CI. (c) Correlation analysis between signals (mean ± SEM) of positive samples versus immune-informatics predicted epitope density (median ± range) for each recombinant protein.
Mentions: The following antigens (Ags) were employed: mannosylated lipoarabinomannan (ManLAM) from H37Rv strain (kindly provided by J. Belisle, Colorado State University, Fort Collins, CO, USA); protein purified derivative (PPD) (Statens Serum Institut, Copenhagen, Denmark). Homemade indirect enzyme-linked immunosorbent assay (ELISA) was used for detection of serum Abs employing goat anti-human polyvalent (anti-IgA/IgM/IgG) horseradish peroxidase- (HRP-) conjugated secondary Ab as detection reagent (Sigma-Aldrich Co. St. Louis, MO, USA). Briefly, 96-well high binding microplates (Corning, USA) were coated overnight (ON) at 4°C with 100 μL of the following Ags: PPD (1 μg/mL, in CO3 buffer pH = 9), ManLAM (0.5 μg/mL, in PO2 buffer pH = 8), and recombinant proteins (1 μg/mL, in PBS pH = 7.4). Optimal Ag concentration and coating buffer was experimentally determined (37, and data not shown). After properly washing with PBS 0.05% (v/v) tween-20 (Sigma) and blocking with PBS 2% (v/v) human serum albumin (HSA) (NatoCor, Córdoba, Argentina) plates were seeded and ON incubated at 4°C with serum dilutions: 1/1000 (PPD), 1/800 (ManLAM), and 1/500 (recombinant proteins). Serum dilutions were established in preliminary assays. Each sample was tested in duplicate. In assays with recombinant proteins (Figure 3), unspecific Abs were depleted from sera by adsorption on plates coated with glutathione-S-transferase (GST) (1 μg/mL, in PBS pH = 7.4) for 1 h at 37°C before serum-Ag incubation step. To deplete Abs directed to surface exposed Mtb-Ags from sera samples (Figure 5(b)), 150 μL of 1/500 diluted sera was also preadsorbed with 106  γ-irradiated H12435 or LAM10406 clinical isolates for 1 h at 37°C before serum-Ag incubation step. Finally, secondary reagent (100 μL, 1/3000 dilution) was added for 30 min and, after extensive washing, HRP activity was measured using tetramethylbenzidine (TMB) substrate (Sigma). Absorbance was measured at 450 nm (subtracting 570 nm background signal) using an UVM340 plate reader with DigiRead software (Asys Hitech, UK).

Bottom Line: Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response.We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins.Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB.

View Article: PubMed Central - PubMed

Affiliation: IMEX-CONICET, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 CABA, Argentina.

ABSTRACT
Molecular epidemiology has revealed that Mycobacterium tuberculosis (Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was also correlated with anti-Mtb-cell surface IgM, but not with anti-ManLAM, anti-PPD, or anti-Mtb-surface IgG antibodies. Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB.

Show MeSH
Related in: MedlinePlus