Evaluation of T-cell responses to novel RD1- and RD2-encoded Mycobacterium tuberculosis gene products for specific detection of human tuberculosis infection

Abstract

The tuberculin skin test for diagnosing Mycobacterium tuberculosis infection suffers from antigenic cross-reactivity of purified protein derivative with BCG, resulting in poor specificity in BCG-vaccinated populations. Comparative genomics has identified several genetic regions in M. tuberculosis and M. bovis that are deleted in M. bovis BCG. Proteins encoded in these regions will form the basis of new specific T-cell-based blood tests that do not cross-react with BCG, but only two, early secretory antigen target 6 and culture filtrate protein 10, have been studied in detail in humans. We investigated four novel gene products, encoded by RD2 (Rv1989c) and RD1 (Rv3873, Rv3878, and Rv3879c), that are absent from most or all of the vaccine strains of BCG, respectively. Sixty-seven overlapping peptides were tested in ex vivo gamma interferon enzyme-linked immunospot assays in 49 patients with culture-confirmed tuberculosis and 38 healthy BCG-vaccinated donors. Forty-five percent (95% confidence interval [CI], 31 to 57%) and 53% (95% CI, 39 to 67%) of the tuberculosis patients responded to Rv3879c and Rv3873, respectively, identifying these proteins as major M. tuberculosis T-cell antigens in humans, while 35 and 25% of the patients responded to Rv3878 and Rv1989c, respectively. Of the 38 BCG-vaccinated donors, 1 (2.6%) responded to peptides from Rv3878 and Rv3879c, 3 (7.9%) responded to Rv3873, and none responded to Rv1989c. Exclusion of cross-reactive peptides encoded in conserved motifs of Rv3873, a PPE family member, increased its specificity to 97.4%. The high specificity of Rv3879c peptides and nonconserved Rv3873 sequences, together with their moderate sensitivity in tuberculosis patients, identifies these peptides as candidates for inclusion in new T-cell-based tests for M. tuberculosis infection.

FIG. 1.

Proportions of culture-confirmed TB patients (n = 49) and healthy, unexposed BCG vaccinees (n = 38) responding in an IFN-γ ELISPOT assay to peptide pools from the four RD region gene products. PBMC from each participant were tested by the IFN-γ ELISPOT assay with peptide pools of between five and seven peptides representing different antigens from RD1 (Rv3873, Rv3878, and Rv3879c) and RD2 (Rv1989c). (A) Percentages of culture-confirmed TB patients and unexposed BCG vaccinees who responded to each peptide pool in an IFN-γ ELISPOT assay. (B) Percentages of culture-confirmed TB patients and unexposed BCG vaccinees who responded to one or more peptide pools from each of the RD1 and RD2 gene products. Total, percentage of donors who responded to 1 or more of the 11 peptide pools from the four antigens PPD, percentage of donors who responded to PPD; solid columns, response rates of TB patients; hatched columns, response rates of unexposed, BCG-vaccinated donors.

FIG. 2.

Magnitudes of IFN-γ ELISPOT assay responses to RD region antigens in 49 culture-confirmed TB patients (A) and 38 healthy, unexposed BCG vaccinees (B). Frequencies of peptide-specific IFN-γ-secreting SFC summated for each of the constituent peptide pools for each antigen, enumerated by ex vivo ELISPOT assay in patients with TB (A) and healthy, unexposed, BCG-vaccinated donors (B), are shown. Each horizontal bar represents the median response for each antigen. Points on the baseline represent individuals with no response to a given antigen (i.e., fewer than five SFC above the negative control for each of the constituent peptides of each pool of the given antigen). The broken horizontal line represents the predefined cutoff point (five SFC per 2.5 × 10⁵ PBMC, which translates into a threshold of detection of 20 peptide-specific T cells per 10⁶ PBMC).

FIG. 3.

Location and homology of PPE protein family motif (as described at http://genolist.pasteur.fr/TubercuLIST/mast/P210.1.html) within the partial amino acid sequence of Rv3873 (amino acid residues 100 to 160). Amino acid residues are shown in the one-letter code. Underlined residues indicate the given peptide sequence. Identical residues are indicated with a cross.