Monitoring tuberculosis progression using MRI and stereology

Transcription

1 Monitoring tuberculosis progression using MRI and stereology

2 TB the problem Estimated number of new cases in million deaths; 9 million new cases p.a. TB kills someone every 15 secs, 9,153 cases in UK (2009), Latent infection (one third of world s population) Co-infection with HIV, drug resistance (MDR, XDR)

3 Mycobacterium tuberculosis - antibiotic therapy Drug sensitive strains: 6+ months therapy: 2-4 antibiotics 5k Drug resistant strains: 18+ months therapy: 5+ antibiotics 50-70k UK = 70 million pa Issues: - non-compliance - side-effects Increasing numbers of MDR / XDR strains

4 Mycobacterium tuberculosis - vaccination BCG vaccine M. bovis [bacille Calmette Guerin]: live, attenuated inexpensive, safe, 3 billion doses effective in children - not adults variable efficacy globally [0-80%] live vaccine not recommended for babies with HIV does not prevent infection [latency]

5 Background The global needs: Therapy - new drug targets - shorter treatment times Vaccines - improve BCG / boost BCG (sub-unit vaccine) - NO CURRENT CORRELATE OF PROTECTION In vivo Models - Well characterised - Predictive - Clear readouts

6 Animal models of M. tuberculosis infection Rational approach to model development and use Three species predominantly used for efficacy studies Vaccines SUSCEPTIBILITY move through TO INFECTION progressively WITH more M. tuberculosis complex models prior to clinical trial Clinical Trials - Immunogenicity, mechanisms of action - Protection against Mtb challenge - Protection against Mtb challenge, - more stringent & discriminative - Detailed and relevant immunogenicity - Safety - Protection against Mtb challenge

7 Macaque models for TB vaccine evaluation 2 species are commonly used to model human tuberculosis Rhesus macaque Cynomolgus macaque Macaca mulatta mulatta: Indian and Chinese subspecies Macaca fascicularis: Native: e.g. S.E. Asia, Philippines. Introduced alien species: e.g. Mauritius. History : Rhesus macaque; aerosol / intra-bronchial challenge; BCG effective 1996 to date: Cynomolgus macaque: intra-tracheal challenge 2001: Langermans et al: BCG protective in cynomolgus, partially protective in rhesus. Lack of clear criteria for species selection for the evaluation of therapeutic interventions against tuberculosis. Need for further studies to: Characterise the species and the model for different uses. Define and improve study endpoints and readouts.

8 Aims To establish aerosol challenge models of TB infection in NHP To characterise and compare rhesus and cynomolgus macaque species as models of human tuberculosis for vaccine assessment. Susceptibility to infection with M. tuberculosis Similarity to humans To evaluate readouts of vaccine immunogenicity and efficacy including the evaluation of advanced imaging approaches to enhance measurement of disease burden

9 Aims To establish aerosol challenge models of TB infection in NHP To characterise and compare rhesus and cynomolgus macaque species as models of human tuberculosis for vaccine assessment. Susceptibility to infection with M. tuberculosis Similarity to humans To evaluate readouts of vaccine immunogenicity and efficacy including the evaluation of advanced imaging approaches to enhance measurement of disease burden

10 Aerosol Challenge Model Clinical parameters Humane endpoint indicators Behavioural indicators Depressed or withdrawn behaviour, Abnormal respiratory rates (dyspnoea). Weight Loss of 20% of peak post-challenge weight. Temperature Increased temperature Measures of inflammation Erythrocyte Sedimentation Rate ESR levels > normal Chest X-ray Severely abnormal chest X-ray Haematology: Full blood count (CBC) Hb Haemoglobin level < normal limits X-radiograph

11 Incubation (Days). Incubation (days). Incubation (days). Incubation (days). Aerosol Challenge Model Assessment of the Immune response Identification of correlates of protection, disease pathogenesis - Time course studies Immune responses investigated: Cytokine IFN-γ model calibrator; ELISPOT, ELISA, ICS Cellular responses: e.g. Polyfunctional T cells, proliferation Mucosal responses: BAL, lung Humoral responses Omics Mean Conc pg/ml IFNg Mean Conc pg/ml Mucosal MVA85A vaccination group Intradermal MVA85A vaccination group TNFa. Mucosal T-cell profile (BAL) Peripheral T-cell profile (PBMC) ELISpot method Aligned with human clinical trials ELISpot method ELISPOT ELISPOT: measures Frequency the IFN-γ Frequency secreting of cells specific cells ELISA: which Quantity secrete of IFN-γ secreted Anti-IFNgamma Cells IFN- γ Detection complex coating antibody (mononuclear Anti-IFNgamma cells (biotinylated Cells detector IFN- antibody, γ Detection complex isolated coating from antibody blood or lymphoid tissue) (mononuclear Streptavidin-ALP cells isolated and from BCIP/NBT blood substrate) (biotinylated detector antibody, Streptavidin-ALP or lymphoid tissue) and BCIP/NBT substrate) ELISA measures the Quantity of IFN-γ s PPD-specific IFN-gamma producing cells/10 6 PBMC 100μl 100μl 500 1:5 diluted blood Antigen solution Rhesus medians (n=12) Human median values (n=8) days at 37 o C Supernatants stored at 80 o C pre Weeks post-bcg IFN-gamma vaccination ELISA assay Mean Conc pg/ml. IL-2 IL Mean Conc pg/ml

12 Aerosol Challenge Model Assessment of disease at necropsy Pathology / Histology Scoring system for TB NHP disease pathology (qualitative) Pulmonary disease Pathology score Manual lesion count H&E for evaluation of lesion pathology Bacteriology Organ-specific bacterial load Evaluation of imaging for determination of pulmonary disease Visualisation of disease Quantification of disease (lesion counting; lesion : lung volume by stereology)

13 Aerosol Challenge Model development VACCINE EVALUATION AEROSOL CHALLENGE DOSE ESTABLISHED READOUTS OF DISEASE BURDEN ESTABLISHED IMMUNE ASSAYS ESTABLISHED ALIGNED HUMAN CLINICAL TRIALS HUMANE ENDPOINT CRITERIA ESTABLISHED X-RAY CAPABILITY HOUSING SYSTEM - CL3 CONTAIMENT AEROSOL DELIVERY SYSTEM: NHP PLETHYSMOGRAPHY

14 Readouts of disease burden for evaluation of TB vaccine efficacy Vaccine evaluation studies require readouts capable of demonstrating that vaccine candidates are able to protect against challenge with tuberculosis. The ideal readout would be Discriminatory Sensitive Objective Quantitative Transferable

15 % survival Readouts of disease burden for evaluation of TB vaccine efficacy Current readouts Survival: Long term studies required, larger numbers. Clinical assessment: X-ray: Qualitative, not easily transferable. Gross pathology: Qualitative, not easily transferable. Bacterial load in tissues: Technically challenging. Test groups with survivors and non survivors show a large variation in group bacterial load. D53: Pre exposure D53: 4 weeks post exposure to Mtb Immune responses: No correlate of protection Survival time post-challenge Time post-challenge (days) BCG prime, MVA85 boost (id) BCG prime, MVA85 boost (in) BCG prime, Ad85 boost (in) BCG prime, Rv3407 boost BCG::2F9 EsxA- His BCG Danish Saline Viable M. tuberculosis Log10 CFU/ml Lung Spleen < <0.001 BCG Danish (1331) s.c Lipid Pk negative control BCG Moreau oral BCG Danish Lipid Pk oral BCG Moreau Lipid Pk oral Viable M. tuberculosis Log 10 CFU/ml BCG Danish (1331) s.c Lipid Pk negative control BCG Moreau oral BCG Danish Lipid Pk oral BCG Moreau Lipid Pk oral Survival Clinical assessment Bacterial load

16 Improved readouts - Advanced Medical Imaging Imaging systems Computed tomography (CT) uses X-rays to construct good high resolution 3D images of tissue. Shorter scan time, less detailed images, bone imaging possible Magnetic resonance imaging (MRI) uses strong magnetic fields and nonionising radiation to generate high contrast 3D images Longer scan time, high resolution images, multiple scan types, not calcification MR Imaging of TB infected lungs performed ex-vivo Lungs inflated with formalin, embedded in agarose Contained samples transported to Oxford for scanning (MR, CT) Visualisation of disease burden Quantification of disease burden CT - MR stereology Total lung and lesion volumes determined from MR digital image stacks Lesion : lung volume ratio determined as measure of disease burden

17 Imaging based readouts of disease burden Visualisation of pulmonary disease: Pathology Rhesus macaques Dose M. tuberculosis High: 75 CFU Mid: 45 CFU Low: 30 CFU Pulmonary disease induced following aerosol exposure. Increased level of disease burden with increased exposure dose of M. tuberculosis.

18 Imaging based readouts of disease burden Visualisation of pulmonary disease: MR Images Rhesus macaques Dose M. tuberculosis 75 CFU 40 CFU 30 CFU Even distribution of disease following aerosol exposure. Clearly shows the increased level of disease burden with increased exposure dose of M. tuberculosis.

19 Imaging based readouts of disease burden Visualisation of pulmonary disease: MR Images Rhesus Mauritian Chinese Cynomolgus Cynomolgus Increased level of disease burden in Rhesus and Mauritian cynomolgus macaques compared to Chinese Cynomolgus macaque

20 Total lesion count Imaging based readouts of disease burden Quantification of pulmonary disease Study 2.1: Comparison of total lung lesion counts obtained from MRI scans and Lesion by slicing and counts manual counting Order of challenge Rhesus macaques CT MR Manual Lesions enumerated from Cross sectional CT or MR images Sliced lung sections Number of lesions D53 D31 D80 Dose M. tuberculosis CFU X-ray Score Excellent correlation of CT, MR, with pathology counts. Counting provides an over all view of disease burden Limitations at high levels of disease Lesion size not included

21 Lesion : lung volume Imaging based readouts of disease burden Quantification of pulmonary disease Lesion volume : Lung volume [MR stereology] Sharpe et al., Tuberculosis 0.4 Rhesus macaques Rhesus macaques D53 D12 D19 D28 D42 C54 D60 D80 D31 Dose Dose M. tuberculosis CFU The lesion to lung ratio is an indicator of disease severity, the higher the ratio the greater the proportion of lung is taken up with lesions. ratio

22 M. tuberculosis infection studies Disease burden measures: 0.4 lesion volume: lung volume ratio (MRI) Rhesus macaques 0.3 Quantitative measures Lesion volume / Lung volume ratio Lesion: lung volume Lesion count Number in lung Total lung lesion count D53 D12 D19 D28 D42 C54 D60 D80 D31 Qualitative measures Lung gross path score Total gross path score Score Score Lung pathology score Total pathology score D53 D12 D19 D28 D42 C54 D60 D80 D31 D53: Pre exposure D53: 4 weeks post exposure to Mtb Blue fill indicates animals which met humane endpoint before week 12 Lesion : lung volume ratio correlates with the other measures of pulmonary disease assessed Chest X-ray score Score Chest X ray Score D53 D12 D19 D28 D42 C54 D60 D80 D31 Mtb Challenge to euthanise interval (weeks) Quantitative data was more sensitive than the Qualitative measures of pulmonary disease (gross pathology score, Chest X-ray score).

23 Lesion : lung volume Lesion volume : Lung volume [MR stereology] Vaccine efficacy readout Sharpe et al., Clinical & vaccine Immunology 0.4 Unvaccinated 0.4 BCG vaccinated k54 K47 K62 K K44 K52 K80 K43 K50 K59 Grey fills indicates animals which did not met humane endpoint before end of study (week 52) A significant difference lesion volume to lung volume ratio was seen between the unvaccinated animals and animals vaccinated with BCG alone (p = 0.011)

24 In life imaging Use of mobile scanner units MR: Abdomen apex to base CT: Head to base

25 In life imaging at HPA Porton CT imaging with software reconstruction CT: voyage down the trachea Bronchoscopy: Trachea

26 Summary Visualisation of pulmonary disease - MR imaging provided a clear indication of pulmonary disease burden in fixed tissues. Quantification of disease burden: Lesion counting - Provided an overall view of pulmonary disease burden, but didn t take lesion size into account. - Agreement between lesion counts from CT or MR images with counts from pathological specimens validates the approach for use in life. MR stereology (lesion volume to lung volume ratio) - Provides a tool to quantify pulmonary disease induced by M. tuberculosis infection. - Superior to qualitative readouts of disease burden. - Provides a reliable measure against which to validate the Immunological correlates of vaccine-induced protective efficacy.

27 Future - Imaging Inclusion of imaging in future studies Application of stereology approach to CT image stacks In life imaging Development of a suitable containment strategy Evaluation of MR and CT modalities Evaluation of PET CT