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Pilot study of diagnostic potential of the Mycobacterium tuberculosis recombinant HBHA protein in a vaccinated population in Finland.

Savolainen L, Pusa L, Kim HJ, Sillanpää H, Seppälä I, Tuuminen T - PLoS ONE (2008)

Bottom Line: At present these assays do not discriminate between disease and latency.The T cell reactivities to HBHA were compared to the respective reactivities towards Purified Protein Derivative (PPD) and two surface secreted proteins, ie.Our pilot results indicate that methylated recombinant HBHA induced a strong T cell mediated immune response and the production of IgG and IgM-class antibodies in all patient groups, most surprisingly in young Finnish vaccinees, as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland.

ABSTRACT

Background: In recent years T cell based interferon gamma release assays (IGRA) have been developed for immunodiagnosis of M. tuberculosis infection. At present these assays do not discriminate between disease and latency. Therefore, more promising antigens and diagnostic tools are continuously being searched for tuberculosis immunodiagnostics. The heparin binding hemagglutinin (HBHA) is a surface protein of M. tuberculosis which promotes bacterial aggregation and adhesion to non-phagocytic cells. It has been previously assumed that native, methylated form of this protein would be a promising antigen to discriminate latent from active infection.

Methodology and principal findings: We performed a pilot investigation to study humoral and T-cell mediated immunological responses to recombinant HBHA produced in M. smegmatis or to synthetic peptides in patients with recent or past tuberculosis, with atypical mycobacteriosis, or in healthy vaccinated individuals. The T cell reactivities to HBHA were compared to the respective reactivities towards Purified Protein Derivative (PPD) and two surface secreted proteins, ie. Early Secretory Antigen Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10). Our pilot results indicate that methylated recombinant HBHA induced a strong T cell mediated immune response and the production of IgG and IgM-class antibodies in all patient groups, most surprisingly in young Finnish vaccinees, as well. We observed a positive correlation between the reactivities to HBHA and non-specific PPD among all studied subjects. As expected, ESAT-6 and CFP-10 were the most powerful antigens to discriminate disease from immunity caused by vaccination.

Conclusions: On the basis of results of this exploratory investigation we raise concerns that in countries like Finland, where BCG vaccination was routinely used, HBHA utility might not be sufficient for diagnostics because of inability to explicitly discriminate tuberculosis infection from immunoreactivity caused by previous BCG vaccination.

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Related in: MedlinePlus

The serological reactivities to synthetic peptides, rMtb-HBHA and PPD were tested in IgG and IgM ELISAs.The groups of patients with inactive TB (n = 9), active TB (n = 4) and healthy vaccinated individuals (n = 4) were enrolled. Serological responses to rMtb-HBHA measured by the IgG EIA (A), IgM EIA (B), and IgG correlation in the PPD and rMtb-HBHA ELISAs (C). IgM responses to methylated 15-mer linear peptides from the C terminus of HBHA: IELPKKAAPA[KMe]KAAP (D), AAPAKKAAPA[KMe]KAAA (E), and AAPAKKAAPA[KMe][KMe]AAA (F). Individual responses are presented as optical densities (OD405). The horizontal bars represent arithmetic median values.
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pone-0003272-g003: The serological reactivities to synthetic peptides, rMtb-HBHA and PPD were tested in IgG and IgM ELISAs.The groups of patients with inactive TB (n = 9), active TB (n = 4) and healthy vaccinated individuals (n = 4) were enrolled. Serological responses to rMtb-HBHA measured by the IgG EIA (A), IgM EIA (B), and IgG correlation in the PPD and rMtb-HBHA ELISAs (C). IgM responses to methylated 15-mer linear peptides from the C terminus of HBHA: IELPKKAAPA[KMe]KAAP (D), AAPAKKAAPA[KMe]KAAA (E), and AAPAKKAAPA[KMe][KMe]AAA (F). Individual responses are presented as optical densities (OD405). The horizontal bars represent arithmetic median values.

Mentions: Twenty three 15-mer sequential peptides overlapping by nine amino acids and spanning the genomic sequence of the M. tuberculosis (H37Rv) HBHA-protein starting from 37th amino acid were synthesized (Proimmune, Oxford, UK; Alta Bioscience, Birmingham, UK). The average purity of the peptides was ∼73%. Three peptides from the protein C-terminus were chemically methylated at their lysine residues (see Legend of Fig. 3). The methylation was done on the PEPscreen synthesis platform. Fmoc-Lys(Me,Boc)-OH were pre-dissolved at a 0.5 M concentration, placed on the deck, coupled and deprotected in the same way as standard amino acids. Additionally, the peptides for serology were biotinylated at their N-terminus. The quality control for all peptides was accomplished by mass spectrometry analysis. The peptides were dissolved in sterile asetonitrile and stored at −20°C in aliquots of 1–4 mg/ml with ∼30% glycerol for serology and in PBS for the T-cell assays.


Pilot study of diagnostic potential of the Mycobacterium tuberculosis recombinant HBHA protein in a vaccinated population in Finland.

Savolainen L, Pusa L, Kim HJ, Sillanpää H, Seppälä I, Tuuminen T - PLoS ONE (2008)

The serological reactivities to synthetic peptides, rMtb-HBHA and PPD were tested in IgG and IgM ELISAs.The groups of patients with inactive TB (n = 9), active TB (n = 4) and healthy vaccinated individuals (n = 4) were enrolled. Serological responses to rMtb-HBHA measured by the IgG EIA (A), IgM EIA (B), and IgG correlation in the PPD and rMtb-HBHA ELISAs (C). IgM responses to methylated 15-mer linear peptides from the C terminus of HBHA: IELPKKAAPA[KMe]KAAP (D), AAPAKKAAPA[KMe]KAAA (E), and AAPAKKAAPA[KMe][KMe]AAA (F). Individual responses are presented as optical densities (OD405). The horizontal bars represent arithmetic median values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2533402&req=5

pone-0003272-g003: The serological reactivities to synthetic peptides, rMtb-HBHA and PPD were tested in IgG and IgM ELISAs.The groups of patients with inactive TB (n = 9), active TB (n = 4) and healthy vaccinated individuals (n = 4) were enrolled. Serological responses to rMtb-HBHA measured by the IgG EIA (A), IgM EIA (B), and IgG correlation in the PPD and rMtb-HBHA ELISAs (C). IgM responses to methylated 15-mer linear peptides from the C terminus of HBHA: IELPKKAAPA[KMe]KAAP (D), AAPAKKAAPA[KMe]KAAA (E), and AAPAKKAAPA[KMe][KMe]AAA (F). Individual responses are presented as optical densities (OD405). The horizontal bars represent arithmetic median values.
Mentions: Twenty three 15-mer sequential peptides overlapping by nine amino acids and spanning the genomic sequence of the M. tuberculosis (H37Rv) HBHA-protein starting from 37th amino acid were synthesized (Proimmune, Oxford, UK; Alta Bioscience, Birmingham, UK). The average purity of the peptides was ∼73%. Three peptides from the protein C-terminus were chemically methylated at their lysine residues (see Legend of Fig. 3). The methylation was done on the PEPscreen synthesis platform. Fmoc-Lys(Me,Boc)-OH were pre-dissolved at a 0.5 M concentration, placed on the deck, coupled and deprotected in the same way as standard amino acids. Additionally, the peptides for serology were biotinylated at their N-terminus. The quality control for all peptides was accomplished by mass spectrometry analysis. The peptides were dissolved in sterile asetonitrile and stored at −20°C in aliquots of 1–4 mg/ml with ∼30% glycerol for serology and in PBS for the T-cell assays.

Bottom Line: At present these assays do not discriminate between disease and latency.The T cell reactivities to HBHA were compared to the respective reactivities towards Purified Protein Derivative (PPD) and two surface secreted proteins, ie.Our pilot results indicate that methylated recombinant HBHA induced a strong T cell mediated immune response and the production of IgG and IgM-class antibodies in all patient groups, most surprisingly in young Finnish vaccinees, as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland.

ABSTRACT

Background: In recent years T cell based interferon gamma release assays (IGRA) have been developed for immunodiagnosis of M. tuberculosis infection. At present these assays do not discriminate between disease and latency. Therefore, more promising antigens and diagnostic tools are continuously being searched for tuberculosis immunodiagnostics. The heparin binding hemagglutinin (HBHA) is a surface protein of M. tuberculosis which promotes bacterial aggregation and adhesion to non-phagocytic cells. It has been previously assumed that native, methylated form of this protein would be a promising antigen to discriminate latent from active infection.

Methodology and principal findings: We performed a pilot investigation to study humoral and T-cell mediated immunological responses to recombinant HBHA produced in M. smegmatis or to synthetic peptides in patients with recent or past tuberculosis, with atypical mycobacteriosis, or in healthy vaccinated individuals. The T cell reactivities to HBHA were compared to the respective reactivities towards Purified Protein Derivative (PPD) and two surface secreted proteins, ie. Early Secretory Antigen Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10). Our pilot results indicate that methylated recombinant HBHA induced a strong T cell mediated immune response and the production of IgG and IgM-class antibodies in all patient groups, most surprisingly in young Finnish vaccinees, as well. We observed a positive correlation between the reactivities to HBHA and non-specific PPD among all studied subjects. As expected, ESAT-6 and CFP-10 were the most powerful antigens to discriminate disease from immunity caused by vaccination.

Conclusions: On the basis of results of this exploratory investigation we raise concerns that in countries like Finland, where BCG vaccination was routinely used, HBHA utility might not be sufficient for diagnostics because of inability to explicitly discriminate tuberculosis infection from immunoreactivity caused by previous BCG vaccination.

Show MeSH
Related in: MedlinePlus