Population tailored modification of tuberculosis specific interferon-gamma release assay

Abstract

Objectives: Blood-based Interferon-Gamma Release Assays (IGRA) identify Mycobacterium tuberculosis (MTB) sensitisation with increased specificity, but sensitivity remains impaired in human immunodeficiency virus (HIV) infected persons. The QuantiFERON-TB Gold In-Tube test contains peptide 38-55 of Rv2654c, based on data indicating differential recognition between tuberculosis patients and BCG vaccinated controls in Europe. We aimed to fine map the T cell response to Rv2654c with the view of improving sensitivity.

Methods: Interferon-gamma ELISpot assay was used in HIV uninfected persons with latent and active tuberculosis to map peptide epitopes of Rv2654c. A modified IGRA was tested in two further groups of 55 HIV uninfected and 44 HIV infected persons, recruited in South Africa.

Results: The most prominently recognised peptide was between amino acids 51-65. Using p51-65 to boost the QuantiFERON-TB Gold In-Tube assay, the quantitative performance of the modified IGRA increased from 1.83 IU/ml (IQR 0.30-7.35) to 2.83 (IQR 0.28-12.2; p = 0.002) in the HIV uninfected group. In the HIV infected cohort the percentage of positive responders increased from 57% to 64% but only after 3 months of ART (p = ns).

Conclusions: Our data shows the potential to population tailor detection of MTB sensitization using specific synthetic peptides and interferon-gamma release in vitro.

Keywords: HIV; Interferon-Gamma Release Assays (IGRA); Peptide epitope; QuantiFERON assay; Rv2654c; Tuberculosis.

Figure 1

Identification of highly immunogenic regions by MHC-binding predictions. The median value of the consensus percentile rank scores of binding to supertype-defining HLA II alleles was obtained from predictions by the IEDB webservice, for each 15-mer peptides. Since the smaller consensus percentile rank a peptide has, the better binding is expected, percentile rank values were converted to binding scores by subtraction of the value from 100. The 100 minus median rank values are shown for the top scoring 20% (15 peptides) as a function of starting residues.

Figure 2

Detailed analysis of the C-terminal 51–81 region of Rv2654c compared to p38-55 peptide. The number of spot forming cells per million PBMC (SFC/10⁶) are presented from n = 19 MTB sensitized individuals. The cut-off for positive response was 20 SFC/10⁶ above nil (dotted line). Significance was calculated using Mann–Whitney U test. **p < 0.005. Black lines represent the median SFC/10⁶ values on both panels.

Figure 3

Population tailored epitope prediction. The portion of the population able to present a 15-mer peptide was estimated based on simulations combining MHC binding scores of the IEDB epitope prediction tool and HLA serotype frequencies described in the Xhosa population. The 5 best scoring peptides for a given combination of HLA alleles were regarded as binders (binding to an APC), with a fair chance for antigen presentation. The number of people likely to have a given combination of HLA alleles was estimated using the Hardy–Weinberg equation. Percentage of the whole population, expected to present a given peptide is given for each possible starting position of 15-mers derived from the Rv2654 protein. Panel (A) represents the percentage of the Xhosa population, expected to present 15-mer peptides, based on both HLA-DR and HLA-DQ predictions of the IEDB tool. On panel (B), percentage of Xhosa population based only on HLA-DR prediction is presented, while on panel (C), percentage of the Danish population, based on HLA-DR predictions is represented.