Abstract
Purpose :
Intra-ocular tuberculosis (IOTB) is a significant cause of visual morbidity in TB-endemic regions. Clinically, IOTB mimics non-TB uveitis and being paucibacillary, is difficult to diagnose by bacteriological methods, e.g., M. tb cultures and molecular tests. Most IOTB suspects are given empirical TB treatment, resulting in overtreatment and undertreatment. Studies were conducted to identify M. tb antigens (Ag) and/or antibodies (Ab) to them with the potential of developing diagnostic biomarkers for IOTB.
Methods :
Peptide arrays bearing 15mer peptides (7 aa overlap) of 7 select M. tb Ags (Rv1965, Rv1971, Rv2351c, Rv2675c, Rv3121, Rv1837c and Rv3874) were screened with IO fluid (IOF) from 8 confirmed IOTB patients and 4 non-IOTB controls to identify immunodominant (ID) peptides recognized exclusively by ≥75% (≥6/8) patients. To identify additional Ags potentially expressed in the IO environment, M. tb whole genome microarrays were screened using RNA from M. tb grown in artificial IOF (AIOF) (6 days) and RNA from the log-phase inoculum grown in Middlebrook media as reference; transcripts upregulated ≥ 2 fold were identified.
Results :
34 ID epitopes from 7 proteins exhibited Ab reactivity in ≥75% patient IOF. Replication of M. tb H37Rv in AIOF is markedly reduced, yet the M. tb transciptome at day 6 in AIOF indicated downregulation of the DevR (DosR) regulon which suggests suppression of dormancy. There was upregulation of the Rv2661c-Rv2663 locus (2.7-3.6 fold) and adjacent genes, Rv2660c and Rv2664, were upregulated at earlier time points in supporting experiments. This locus is induced by starvation and associated with hypoxia-induced non-replicating persistence. Recombinant Rv2660c protein is shown to augment expression of pro-inflammatory cytokines IL-1β, IL-8, TNF-α, and IL-12p70, from macrophages; cytokines which are essential for granuloma formation and maintenance. In addition, transcripts for Rv2875 (MPT70), and its predicted functional partner, Rv2876, were upregulated 3 fold. Abs to MPB70 are useful markers for TB in cattle.
Conclusions :
ID epitopes in the 7 Ags recognized by Abs in IOF from IOTB patients are identified. The transcriptome of M. tb replicating in AIOF provides insight into the adaptation of M. tb in the IO environment and may yield additional potential candidates for developing a rapid diagnosis for IOTB.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.