CTL immunogenicity of Rv3615c antigen and diagnostic performances of an ESAT-6/CFP-10/Rv3615c antigen cocktail for Mycobacterium tuberculosis infection

Abstract

T cell immune responses have played pivotal roles in host immune protection against Mycobacterium tuberculosis (MTB) infection. MTB specific antigen, Rv3615c (EspC), was identified to be as immunodominant as the well-known ESAT-6 and CFP-10, and has brought promising expectations to more sensitive T-cell based diagnosis and vaccine development. However, limited knowledge about the immunogenicity and diagnostic values of this antigen has restricted its application in clinical practice. Herein, the Rv3615c antigen was identified as a robust CTL immunoantigen with broadly cross-human leucocyte antigen (HLA) allele recognized peptides which may contribute to the broad recognition of Rv3615c antigen among the population. A three-antigen-cocktail (3-Ag-cocktail) comprising of ESAT-6, CFP-10 and Rv3615c was investigated in a multicenter, randomized and double-blinded study to evaluate its clinical diagnostic performances. A significantly improved sensitivity was demonstrated against the 3-Ag-cocktail compared with that against ESAT-6 and CFP-10. Both responsive magnitude and sensitivity were significantly lower in patients concurrently suffering from cancer, indicating its restriction in diagnosis of immunocomprised patients. In conclusion, inclusion of the Rv3615c antigen with multiple HLA restricted CTL epitopes would benefit the T-cell based diagnosis of MTB infection.

Introduction

According to the report of World Health Organization (WHO) in 2016, tuberculosis (TB) remains a major threat to public health with an estimated 10.4 million new TB cases and 1.4 million TB deaths worldwide in 2015 [1], [2], [3], [4], [5]. One potential concern is latent TB infection (LTBI), in which the MTB bacteria stay inactive while the host remains asymptomatic. It is estimated that the global burden of LTBI is 23% with South-East Asia, Western Pacific and Africa accounted for 80% of these LTBIs [2]. Each LTBI case carries a 5%–10% lifetime risk of progressing to active TB (ATB) disease under certain circumstances such as immune suppression, co-infection with HIV, etc. [1]. Moreover, an ATB patient may cause 10-20 additional infections within a year if left untreated. Large population and heavy disease burden in China has made LTBI a huge reservoir for future ATB prevalence [6]. However, diagnosis of ATB is still difficult and full of uncertainties in clinical practice in the reality of such serious MTB epidemic challenge. Effective diagnosis of MTB infection is also of significance for early therapeutic medications to control disease progression [3]. Microbial culture of smear for acid-fast bacilli test showed poor sensitivity and may have missed a comparable proportion of ATB patients, while radiographic test would usually confounded with other pulmonary diseases [7]. Moreover, for extrapulmonary TB (EPTB) patients, difficulties in sample accession and low bacterial load at the sites of disease have further decreased the sensitivity in bacteriological or pathological diagnosis [8]. Though the old tuberculin skin test has been widely used in screening of MTB infection for more than a century [9], low reactivity in immunocomprised patients and poor specificity caused by Bacillus Calmette-Guerin (BCG) vaccination in part of the world, i.e. programmed BCG vaccination among children throughout China, has restricted its clinical diagnostic values [10], [11].

T-cell responses play pivotal roles in protection against MTB infection and maintenance of immune balance during LTBI [12], [13]. Th1 cell responses have been considered to be the major T-cell responses after MTB infection and are critical in protection against TB from the fact that HIV co-infection, accompanied with the impairment of CD4⁺ T-cell responses, leads to increased susceptibility to MTB [12], [14]. In the meanwhile, mounts of evidence support that CD8⁺ T cells are also needed in protection against TB, especially in LTBI [15], [16], [17]. Previously, efforts in the identification of MTB specific antigens mainly focused on antigens encoded from genes in the region of differences (RD region), which were absent in BCG strains [11]. Detection of T-cell responses against two immunodominant antigens encoded in RD1, i.e. early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), has been widely used in diagnosis of MTB infection [18], [19], [20]. Clinical diagnostic studies with these two antigens demonstrate a high sensitivity of 90%–95% in bacteriologically confirmed patients and the test is not affected by previous BCG vaccination [11], [20].

On the other hand, our understanding of immune protection mechanisms and immunogenicity of MTB during infection is still insufficient, which leaves the identification and investigation of novel immunodominant antigens of great significance for both diagnosis and vaccine development [21], [22]. ESAT-6 system 1 (Esx-1) substrate protein C (EspC; Rv3615c), which is encoded outside RD region but secreted through the RD1 encoded Esx-1secretion system, was defined first in cattle, and then in a small cohort of human patients to be as immunodominant as ESAT-6 and CFP-10 [23], [24]. However, diagnostic performances and immunogenicity mechanisms of this antigen has not yet been well studied, especially when considering the needs for improving diagnostic sensitivities in bacteria-negative and immunocompromised patients (e.g. HIV infection, cancer, etc.), and moreover, for effective vaccine development [25].

In the present study, the CTL immunogenicity mechanisms of Rv3615c antigen was elucidated through the identification of HLA-A restricted peptide clusters among ATB patients. Furthermore, we designed overlapping peptides spanning the whole sequence of widely-applied ESAT-6, CFP-10 antigen and Rv3615c antigen, and mixed them together to constitute a three-antigen-cocktail (3-Ag-cocktail). A multicenter, randomized and double-blinded study was carried out to investigate its clinical diagnostic performance. All these findings would benefit the understanding of immunogenicity mechanisms of MTB and provide useful information for clinical applications of Rv3615c antigen for the diagnosis of MTB infection.