tb research in switzerland: tradition and prospects

Institute of Medical Microbiology

TB research in Switzerland: Tradition and prospects

Peter Sander

Institut für Medizinische Mikrobiologie

Universität Zürich

[email protected]


Content

Mycobacterium tuberculosis and milestones in fight against TB

WHO: Global situation

Research in Switzerland

• Diagnostics

• Drug susceptibility testing, drug resistance mechanisms

• Host pathogen interaction: virulence factors, defense mechanisms

• Epidemiology

• Drug development

• Basic science

• Vaccine

Outlook


Mycobacterium tuberculosis

Gram-positive characteristics of Gram-negative bacteria (MOM)

Impermeable lipid rich cell wall unusual antigens

Slow growing (24 h) culture free identification/resistance testing

Intracellular pathogen phagosome maturation arrest

Low infectious dose

TB: lung (every organ), latent – severe

Therapy: 2 + 4 months (INH, RIF, EMB, PZA) more effective drugs

Development/spread of drug resistant (MDR, XDR) new drugs

Live vaccine: Mycobacterium bovis BCG more effective vaccines


Milestones in Tuberculosis

Lienhardt, … Raviglione (2012) Nat. Rev. Microbiol


WHO – elimination strategies

Dye, …., Raviglione (2012) Annu. Rev. Publ. Health


Tuberculosis Research in Switzerland

Annual TB cases in Switzerland: approx. 550

NCBI/Pubmed (28.02.2013)

Search term: Tuberculosis and Switzerland or Tuberkulose and Schweiz

Hits: 1308

Period (1947- 2012): 1 – 112

SNF database

Search term: Tuberculosis

Projects (1992 – 2015): 47

Applicants/PI: 51

Publications (2007 – 2013): 135


Combat Tuberculosis

Interrupt transmission ! • early detection: clinical diagnosis, laboratory diagnosis

Initiate adequate therapy ! • drug susceptibility testing (phenotypic, genotypic)

Strengthen/prime immune system ! • vaccinate

Develop more potent …. • diagnostics

• drugs

• vaccines

Multidisciplinary research to generate knowledge about host and pathogen !!


Sanatoria


Vitamin D3 – required for IFN-γ-mediated anitmicrobial activity

Science Transl. Med. (2011)


Diagnosis: Clinical marker of active disease

Nat. Med. (2011)


Molecular diagnostics: Detection and identification

Pfyffer, …, Salfinger, Weber (1996) JCM

Implementation of molecular test for non-respiratory specimen

… Bloemberg, Böttger (2010) JCM …. Böttger, 1990, 1996, 2006, 2008


Drug susceptibility testing

Salfinger, Heifets (1988) AAC …. Böttger (2012) JAC

1st line drugs: Pyrazinamide 2nd line drugs: Quinolones


Molecular mechanisms of drug resistance: RifR, EmbR

Telenti, Imboden, ...., Cole, …, Bodmer (1993) Lancet

Alcaide, Pfyffer, Telenti (1997) AAC


Drug resistance mechanisms and MIC

Böttger (2011) Clin. Microbiol. Infect


Epidemiology/Strain typing

Lancet (1993)


Epidemiology - Transmission

520 TB cases (2000 – 2008) 401 foreign born 113 HIV+

72 % Euro-American Mtb lineage 35 clusters (90 patients) Birth origin not associated with clustering HIV+ reduced clustering Most TB patients in CH are foreign born Reduced transmission among HIV-infected patients

JCM (2012)


Strain typing – drug resistance

• Drug resistance mutations associated with various levels of drug resistance and various levels of transmission • Specific Mtb lineages are associated with particular drug-resistance conferring mutations and phenotypic drug resistance

AAC (2012)


Evolution of clinical drug resistance: reconstruction in model organism

… Böttger (2010) Mol. Microbiol.


Evolution of drug resistance/persistence

cytCD (C5) = cytochrome oxidase - impaired in INH tolerance Rv0097 (G4) = unknown ORF - Increased INH tolerance

Dhar, McKinney (2012) PNAS

Stochastic pulsed expression of KatG negatively correlates with survival in the presence of INH

… McKinney (2013) Science


Epidemiology/Evolution of M. tuberculosis – conservation of T cell epitopes

…., Gagneux (2010) Nat. Gen.


Phylogeny of vaccine strain M. bovis BCG: Primary attenuation = deletion of RD1 encoding ESX1


RD1: Virulence related protein secretion system ESX-1

…, Cole, … (2010) JID

…, Cole (2011) J. Bacteriol.


Mtb virulence factors: lipoproteins

Mycobacteria

Sander group (2004, 2007, 2008, 2009, 2010, 2012)

Lung pathology of TB Infected mice


Mtb - Host cell interaction

Mycobacteria retain host cell protein TACO Phagosome maturation arrest

… Pieters (1999) Cell

Pieters (2006) Cell Host Microbe

Mycobacteria secrete eukaryotic like Protein kinase G Phagosome maturation arrest

… Pieters, (2004) Science


Cell/cell spread: M. marinum – Dictyostelium

Hagedorn, … , Soldati (2009) Science


Cellular defense mechanisms

Hagedorn, Soldati (2007) Cell. Microbiol.


Host factors and M. tuberculosis: TNF, Apoptosis, MIF

…, Meylan (1998) J. Immunol. …, Meylan, (2005) Infect. Immun.

MIF = Macrophage migration inhibitory factor


Host factors – interference with apoptosis

…, Meylan (2006) J. Immunol.


Host cell factors: TNFR1, TNF, tm-TNF

Garcia, …. (1997) Eur. J. Immunol. …, Garcia (2002) J. Immunol. …., Garcia (2012) PlosOne

Granuloma formation Wt tg-tm-TNF TNF-/-

Granuloma Wt tg sTNFR1


Host-pathogen interaction: Lipid antigens and presentation by non classically restricted T cells

…, DeLibero (2004) J. Exp. Med. …, DeLibero (2005) Science


Drug development: Benzothiazinones

Target: Decaprenlyphosphoryl-β— D-ribose-2’ epimerase (arabinan synthesis)

…., Cole (2009) Science


Drug development: Pyridomycin

Natural product Target: InhA Drug Active against INH-resistant katG mutants

….Cole (2012) EMBO Mol. Med.


Drug Development: Apramycin

• Structurally unususal aminoglycoside • Licensed for veterinary use • Active against bacteria (MDR) • Little toxicity

Mitochondrial in organello translation

Cochlea explants

Matt, …, Böttger (2012) PNAS

Ctrl.l

Apramycinl

l Gentamicinl


Mycobacterial physiology: Proteasomal degradation

Striebel, …, Weber-Ban (2009) Nat. Struct. Biol. Imkamp, …, Weber-Ban (2011) EMBO Rep. Imkamp, …, Weber-Ban (2011) Mol. Microbiol.


Vaccine improvement: BCG stabilization

Sander, Böttger (2003) Vaccine

Increased stability of BCG recA BCG Russia – a natural recA mutant

Keller, Böttger, Sander (2008) BMC Microbiol


Mycobacterium tuberculosis zmp1

… Sander …Cell Host & Microbe (2008) 3: 224

1. Phagosome maturation:LysoTracker co-localization

2. Increased caspase 1 activation and IL-1β secretion


In-vivo immunogenicity of BCG zmp1 and characterization of enzyme

CD4 CD8

C D P=0.008

P=0.004 wt vs. zmp1 P=0.0476

wt vs. zmp1 P=0.0201 wt vs. zmp1

P<0.0001

Immunisation

…. Sander, ….(2011) J. Biol. Chem. … Böttger, Sander (2011) Clin. Vaccine Immunol. … Sander (2012) Biochem. J.


Improved protective efficacy of BCG zmp in challenge experiments with virulent M. tuberculosis in guinea pigs

BCG zmp BCG Saline3

4

5

6

7

Viab

leM

. tub

ercu

losi

slo

g 10 t

otal

cfu

BCG BCG ∆zmp0

50

100

150

Rel

ativ

e C

FU c

ount

s

Experimental schedule Mo 0: Vaccination 5 x 104 CFU BCG, s.c. Mo 3: Aerosol challenge M. tuberculosis H37Rv 10 – 50 CFU Mo 4: Determination of CFU and lung pathology

Protection of BCG zmp1 in lung significantly and consistently better than BCG (CFU, lung pathology) Improved zmp1 protection independent of genetic background (Pasteur (exp.1), Denmark (exp.2 )) Testing of protective efficacy in non-human primates

LUNG

Sander, Böttger (unpublished)


Tuberculosis research in Switzerland • TB researchers in Switzerland made significant contributions to all fields to combat TB

Diagnostics, drug resistance, drug development, epidemiology, basic science, vaccine development

• TB research in Switzerland is supported by SNF

• TB researchers in Switzerland cooperate at the national and international level

• TB research in Switzerland has capacity to develop synergistic effects

• TB research in Switzerland would benefit from a National Priority Program

• TB research in Switzerland will be required to achieve WHO goal: eradicate TB

Approx 550 TB cases in Switzerland per year (only), but…..


«Eliminating TB by 2050 requires two things: immediate optimization of TB control with the technology we already have, and the concurrent development of a more potent armory of diagnostics, drugs and vaccines.»

«In today’s highly interconnected world, elimination in any country depends on effective TB control in every country.» (WHO report, 2012)


Support

Bonizzzi-Theler Foundation

Institut für Medizinische Mikrobiologie Stiftung wissenschaftliche Forschung UZH


Vaccine development: Protective efficacy of BCG zmp1 in GP

Data: Simon Clark

Protection of BCG zmp1 in lung significantly and consistently better than BCG (CFU, Lung pathology) Improved zmp1 protection independent of genetic background (Pasteur, Denmark)

* 0.0406 * 0.0406

Viab

le M

. tub

ercu

losi

s

(log 1

0 tot

al C

FU)

4

5

6

7

4

5

6

7

Lung

Viab

le M

. tub

ercu

losi

s

(log 1

0 tot

al C

FU)

Lung

Experiment 1 Experiment 2

Vorführender

Präsentationsnotizen

Lung: BCG vs Saline 2.5 log; zmp vs BCG 0.52 log (lung); Spleen: BCG vs. Saline 3.6 log; zmp vs BCG – 0.1 log

tb research in switzerland: tradition and prospects

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