Transcriptomic Signatures Of Recurrent Tuberculosis Disease And Treatment Response In Hiv-Infected Individuals

Table of Contents

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Declaration!............................................................................................................................................................!I!

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Acknowledgements!.......................................................................................................................................!III!

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Summary!..............................................................................................................................................................!V!

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List of abbreviations!...................................................................................................................................!VIII!

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List of Figures!.................................................................................................................................................!XII!

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List of Tables!....................................................................................................................................................!XV!

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List of Equations!...........................................................................................................................................!XVI!

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Chapter 1: Introduction and literature review!.....................................................................................!1!

1.1! From Mtb infection to active TB disease in the absence of immune

suppression!.....................................................................................................................................................!2!

1.1.1 Mtb infection: Epidemiology!..................................................................................................!3!

1.1.2 Diagnosis of Mtb infection!......................................................................................................!4!

1.1.3 Treatment of Mtb infection and prevention of progression to active!................!5!

TB disease!..................................................................................................................................................!5!

1.1.4 Diagnosis of active TB disease!...........................................................................................!8!

1.1.5 Immune response to TB disease!.....................................................................................!10!

1.2 Treatment of active TB disease!................................................................................................!11!

1.2.1 New regimens to shorten TB treatment and prevent relapse.!.........................!12!

1.3 Recurrent TB disease and treatment!.....................................................................................!13!

1.4 HIV and TB co-infection!................................................................................................................!15!

1.4.1 Epidemiology of TB disease and HIV co-infection!......................................................!15!

1.5 Preventing TB in HIV-infected people!....................................................................................!18!

1.6 Progression to active TB, treatment and recurrence in HIV infection!...................!22!

1.6.1 Diagnosis and treatment of active TB disease in HIV-infected persons!.....!23!

1.6.2 Recurrent TB disease in HIV-infected persons!........................................................!24!

1.6.3 HIV immmunopathogenesis and it’s relevance for TB!.........................................!25!

1.7 The search for a biomarker of TB disease and/or protection against TB:

Transcriptomic signatures as biomarkers for TB disease!...................................................!27!

1.8 Biomarkers of TB in HIV co-infection!.....................................................................................!34!

1.9 Biomarkers of risk of TB (prognostic biomarkers)!...........................................................!37!

1.9.1 The ACS 16-gene signature (Zak et al., 2016)!........................................................!40!

1.10 Biomarkers of treatment success in the presence and or absence of HIV

infection!..........................................................................................................................................................!49!

1.10 Biomarkers of risk of recurrent TB disease in the presence and or absence of

HIV infection!................................................................................................................................................!51!

1.11 Type I IFN: Activation and role in active TB disease and HIV infection

pathogenesis!..........................................................................................................................................!51!

1.12 Objectives of this thesis!.........................................................................................................!57!

! XVIII!

Chapter 2: Materials and methods!.......................................................................................................!60!

2.1 Institutional ethics review!..............................................................................................................!60!

2.2: RNA extraction, processing and storage!............................................................................!60!

2.2.1 Extraction from PAXgene tubes!.......................................................................................!60!

2.2.2 RNA extraction from PBMC!...............................................................................................!62!

2.2.3 RNA quantification!..................................................................................................................!63!

2.3 Measurement of gene expression!...........................................................................................!63!

2.3.1 cDNA synthesis and pre-amplification PCR!..............................................................!63!

2.3.2 Fluidigm assay!..........................................................................................................................!65!

2.4 Quality control (QC) of Fluidigm data!.....................................................................................!68!

2.5 Analysis methods!..............................................................................................................................!69!

2.6 Contributions!.......................................................................................................................................!70!

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Chapter 3: The ACS11-gene signature can differentiate active TB from Mtb infection

using PBMC from HIV-infected and uninfected persons!..........................................................!71!

3.1 Introduction!..........................................................................................................................................!71!

3.2 Aim and hypothesis!.........................................................................................................................!74!

Aim:!.............................................................................................................................................................!74!

Hypothesis:!..............................................................................................................................................!75!

3.3 Materials and methods!..................................................................................................................!75!

3.3.1 Ethics clearance and study design!.................................................................................!75!

3.3.2 Sample collection!.....................................................................................................................!77!

3.3.3 RNA extraction and quantification!..................................................................................!77!

3.3.4 Gene expression measurement!.......................................................................................!77!

3.3.5 Data analysis!..............................................................................................................................!78!

3.3.6 Re-parameterisation of the signature to PBMC and HIV infection!................!78!

3.4 Results!....................................................................................................................................................!79!

3.4.1 Cohort characteristics!............................................................................................................!79!

3.4.2: Indistinguishable diagnostic performance of the ACS 11-gene signature in

whole blood and PBMC from HIV-uninfected participants!.............................................!82!

3.4.3: Expression differences of individual signature transcripts and signature

scores in PBMC and whole blood!...............................................................................................!84!

3.4.4: Performance of the signature in diagnosing active TB disease in the HIVinfected

cohort!.......................................................................................................................................!85!

3.4.5: HIV infection upregulates Type I IFN gene expression in whole blood and

PBMC!.........................................................................................................................................................!87!

3.4.6: Development and validation of a modified signature with enhanced

diagnostic performance in HIV infection!..................................................................................!90!

3.5: Discussion!...........................................................................................................................................!94!

3.6: Contributions!......................................................................................................................................!99!

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Chapter 4: Predicting recurrent TB disease in HIV-infected persons on ART!...........!100!

4.1: Introduction!......................................................................................................................................!100!

4.2: Aim and hypothesis!.....................................................................................................................!103!

Aim!............................................................................................................................................................!103!

Hypothesis!............................................................................................................................................!103!

4.3: Methods!.............................................................................................................................................!104!

4.3.1: Study design!..........................................................................................................................!104!

4.3.2: RNA extraction and quantification!..............................................................................!106!

! XIX!

4.3.3: Gene expression measurements!................................................................................!107!

4.3.4: Data analysis!.........................................................................................................................!107!

4.4: Results!...............................................................................................................................................!107!

4.4.1: Participant selection and characteristics!.................................................................!107!

4.4.2: QC of qRT-PCR data!........................................................................................................!113!

4.4.3: Signatures can only predict recurrent TB in the three months preceding

diagnosis in the training set!.........................................................................................................!115!

4.4.4: Performance of transcriptomic signatures in the combined TRuTH training

and test set!...........................................................................................................................................!117!

4.4.5: Lower signature scores in non-progressors than in progressors in the

three months preceding TB recurrence, despite heterogeneity of scores!..........!118!

4.4.8: Signature scores were not associated with time to sputum culture

positivity at recurrent TB diagnosis!.........................................................................................!119!

4.4.9: Time since previous TB diagnosis, time on ART and number of previous

TB episodes were not associated with signature scores!.............................................!119!

4.4.11: The ACS 11-gene and rPSVM.1 signatures could not differentiate Mtb

infection from subclinical TB in ART naïve HIV-infected persons!...........................!122!

4.5: Discussion!........................................................................................................................................!125!

4.6: Contributions!...................................................................................................................................!129!

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Chapter 5: Treatment response monitoring in HIV-infected persons on ART: results

from the IMPRESS cohort!......................................................................................................................!130!

5.1 Introduction!.......................................................................................................................................!130!

5.2: Aims and hypotheses!.................................................................................................................!135!

5.2.1: Aims!............................................................................................................................................!135!

Hypotheses:!.........................................................................................................................................!135!

5.3: Methods!.............................................................................................................................................!136!

5.3.1: Study design!..........................................................................................................................!136!

5.3.2: RNA extraction!......................................................................................................................!137!

5.3.3: Gene expression measurement!..................................................................................!138!

5.3.4: Data analysis!.........................................................................................................................!138!

5.4: Results!...............................................................................................................................................!138!

5.4.1: Participant characteristics and sample stratification!.........................................!138!

5.4.2: QC of qRT-PCR data!........................................................................................................!142!

5.4.3: Signature scores decrease over treatment duration but do not resolve at

the end of TB treatment!.................................................................................................................!144!

5.4.4: Signature scores were not different between early and late converters

during TB treatment.!........................................................................................................................!144!

5.4.5: The ACS 11-gene signature can monitor treatment response in HIVinfected

persons!................................................................................................................................!148!

5.4.6: Early sputum culture conversion is not associated with signature scores

and time to culture positivity at diagnosis!............................................................................!150!

5.5: Discussion!........................................................................................................................................!153!

5.6: Contributions!...................................................................................................................................!157!

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Chapter 6: The effect of HIV viraemia on transcriptomic signature performance in

predicting TB recurrence and monitoring treatment response.!..........................................!158!

6.1: Introduction!......................................................................................................................................!158!

Aim!............................................................................................................................................................!161!

! XX!

Hypothesis!............................................................................................................................................!161!

6.2: Methods!.............................................................................................................................................!161!

6.2.1: Study design!..........................................................................................................................!161!

6.3: Results!...............................................................................................................................................!162!

6.3.1: Stratification of samples in the TRuTH and IMPRESS cohorts for pVL

analysis.!.................................................................................................................................................!162!

6.3.2: HIV viraemia levels are associated with signature scores!.............................!164!

6.3.3: Differentiating between recurrent TB progressors and controls and

monitoring treatment response when HIV pVL is undetectable!...............................!166!

6.4: Discussion!........................................................................................................................................!169!

6.6: Contributions!...................................................................................................................................!170!

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Chapter 7: Signature reduction to pave the way for a point-of-care diagnostic test:

the ACS 6-gene signature!......................................................................................................................!171!

7.1 Introduction!.......................................................................................................................................!171!

7.2: Aims and Hypotheses!................................................................................................................!175!

7.2.1 Aims!.............................................................................................................................................!175!

7.2.2 Hypotheses!..............................................................................................................................!176!

7.3 Methods!..............................................................................................................................................!176!

7.3.1 Study design!............................................................................................................................!176!

7.3.2 Experimental set up and data analyses!....................................................................!177!

7.3 Results!.................................................................................................................................................!178!

7.3.1 Diagnostic performance of the ACS 6-gene signature in HIV-infected and

uninfected persons!...........................................................................................................................!178!

7.3.2 Predicting recurrent TB disease in HIV infected persons on highly active

antiretroviral therapy from the TRuTH cohort!....................................................................!186!

7.3.3 Treatment response monitoring using the ACS 6-gene signature!..............!189!

7.3.4 Effect of plasma viral load levels on ACS 6-gene signature scores in the

TRuTH and IMPRESS cohorts!..................................................................................................!192!

7.4 Discussion!.........................................................................................................................................!194!

7.6: Contributions!...................................................................................................................................!199!

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Chapter 8: General Discussion and Conclusions!......................................................................!200!

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References!.....................................................................................................................................................!209!

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Appendix!.........................................................................................................................................................!245!