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Mycobacterium tuberculosis ¿infectar o matar. Carlos Martin Montañes by murplelake77

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									                 Mycobacterium tuberculosis:
                     ¿infectar o matar?

       “Nuevas Vacunas Contra la Tuberculosis
          de la Investigación al Desarrollo ”



                           Carlos Martín Montañés
http://genmico.unizar.es
                                          1990 MDR TB


                             2006 - 2009 XDR TB


1882 Koch
Mycobacterium tuberculosis

                             1921   Vaccine BCG   1944 Streptomycin
                                                   1951 ISONIAZID
                                                      1972 RIFAMPICIN




                                                                        Modified from Kaufmann PNAS 2005
R Rappuoli Nature Medicine (2004) 10, 1177 - 1185
Evolution Mycobacterium tuberculosis complex




DNA 17.000 years old




     A new evolutionary scenario for the Mycobacterium tuberculosis complex R. Brosch, S. V.
     Gordon, M. Marmiesse, P. Brodin, C. Buchrieser, K. Eiglmeier, T. Garnier, C. Gutierrez, G.
     Hewinson, K. Kremer, L. M. Parsons, A. S. Pym, S. Samper, D. van Soolingen, and S. T. Cole
     Proc Natl Acad Sci 2002 99:3684-9
         M. tuberculosis is a Clonal Population



M. tuberculosis (MTBC)




 M. Canettii
 Smooth tubercle bacilli
                                      Common ancestor of tubercle bacilli species could already have
 M. prototuberculosis                 caused human tuberculosis
 smooth tubercle bacilli
 confined to East Africa
Gutierrez et al PLOS PATHOGENS 2005
                                      Hypothesis that all contemporary humans are derived from an common
                                      ancestor who lived in Africa approximately 300,000 years ago
TODAY GLOBAL DISTRIBUTION M. tuberculosis




 Red Euro-American
                             BEJING IS MOST WORRYING
 Green W-African 1
 Brown W-African 2
 Yellow Indo-Oceanic
 Purple East-Indian
                                           Gagneux et al. PNAS 2006
 Blue East-Asian (Beijing)
VIRULENCE OF SPECIFIC
M. tuberculosis STRAINS?




SUSCEPTIBILITY OF THE
      HOST ?
  NATURAL HISTORY TUBERCULOSIS
                    TB INFECTION   1:3 POPULATION




                                    LATENCY



TB DISEASE (5-10%)
50% Dead without treatment                Nature Medicine 2005
BCG: PRESENT VACCINE
AGAINST TB

Since 1921
LIVE ATTENUATED
VACCINE Mycobacterium                bovis

                                               Albert CALMETTE               Camile GUÉRIN




 EFFECTIVE AGAINST SEVERE FORMS OF CHILDHOOD TB
       Trunz, Fine, Dye. The Lancet 2006, Bonifachich et al. Vaccine 2006.




 BCG MOST USED VACCINE IN THE WORLD

 Protective efficacy againts adult pulmonary TB
 varies greatly between populations !
DISCOVERY STARTED   M. bovis 1908-1921 230 Passages


COMPARATIVE GENOMICS: COMPLEXITY OF BCG VACCINES
BCG Pasteur 1173 SEQUENCE PNAS 2007 Brosch et al

BCG MORE THAN 100 GENES DELETED




   RD1                                     DIFFERENCES IN
ESAT6/CFP10                                IMMUNOGENICITY
                                           DUE TO GENOMIC DIVERSITY?


                                           Group I:
                                           MORE IMMUNOGENIC




                                           Group III:
                                           HIGHLY ATTENUATED
                                                  * Do not produce PDIMs , PGLs
                                                         (Chen et al Vaccine 2007)
RESEARCH ON
NEW TUBERCULOSIS VACCINES



DISCOVERY                      PRECLINICAL             CLINICAL




                           Hundreds of candidates    20 Candidates to 2015
                           Tested in animal models   EU (EDCTP), AERAS…
    1998 Sequence          EU, INH…
 more than 4.000 genes !



   FROM RESEARCH TO                           DEVELOPMENT
                                                        Young and Dye Cell 2006
NEW TB VACCINES:
PUBLIC RESEARCH INITIATIVES: USA




                               I. Orme 2001
               EUROPEAN RESEARCH INITIATIVE
                       EU PROJECTS ON TB VACCINES

"Construction of stable Recombinant BCG and other Live Mycobacterial Vaccines“
EU FP3 BIOTECHNOLOGY CT/92-0520 1993–1996 Coordinator: B. Gicquel I. Pasteur (FR)

"Development of Novel Vaccines by Attenuation of M. tuberculosis"
EU FP4 BIOMED 2 CT/96-2134 1997–1999 Coordinator: B. Gicquel I. Pasteur (FR)


FROM FP5
 "A Cluster for Tuberculosis Vaccine Development"
EU FP5 CT 1999-01093 2000-2004 Coordinator Gicquel I. Pasteur (FR)

TO FP6 TBVAC
“An integrated project for the design and testing of vaccine
candidates against tuberculosis: dentification, Development
and Clinical studies″
EUFP6 2004-2009 Coordinator Jelle Thole Lelystad (NL)

TO TBVI
 RESEARCH ON NEW TB VACCINE
 CANDIDATES
                       DISCOVERY AND PRECLINICAL RESEARCH
                       ON NEW TB VACCINE CANDIDATES

CLUSTER TUBERCULOSIS VACCINE
QLK2-1999-01093 (2000-2004)

SUBUNITS CANDIDATES
   more than 150: identified, produced and tested

LIVE VACCINES
   more than 20: identified, produced and tested

rBCG (2)

Attenuated M. tuberculosis (1) M. tuberculosis phoP
                                                  PRECLINICAL RESEARCH



             TB VAC                               Antigens:
                                                  85 (Oxford, SSI)
                                                  ESAT-6 (SSI)

SUBUNIT                                           Ag Hybrids: 85 + ESAT6
                                                              72f (Glaxo)
VACCINES !
                                                  HBHA: I. Pasteur Lille




                                                      Subunit Vaccine Based
                                                      on a Fusion of Antigen.
                      Nature Structural Biology       Olsen et al. Infect. Immun.2004
                      Rooning et al (2000)
                   PRIME (BCG)                    BOOST                BOOST




Skeiky & Sadoff . Nature Reviews Microbiology 4, 469–476 (June 2006)
      CLINICAL TESTING TB VACCINE

 TB VACCINE                                         STAGE OF                  Pharmaceutical
                       DEFINITION                 DEVELOPMENT                   company or
    TYPE
                                                                              Research Group

SUBUNIT
72f             Selected antigens identified   Phase I trial, ready for         GSK / (EU TB VAC
                from human response            Phase II                             AERAS)
                                               BCG Boosting strategy

85B-ESAT6       Recombinant major antigens     Phase I trial                     SSI (EUTBVAC)
                                               BCG Boosting strategy


VIRAL VECTOR
MVA-85A         Recombinant modified           Phase I , Phase II trials      OXFORD UNIVERSITY
                vaccinia virus Ankara Ag85A    BCG Boosting strategy              (EU TBVAC
                                                                                    AERAS)

LIVE VACCINES
rBCG30          Recombinant BCG: Over          Phase I trial                   (UCLA/INH/ AERAS)
                expression of Ag85B            Prime strategy



                                                    Modified from Curr Opin Pulm Med (2006) Martin C
 LIVE VACCINES TBVAC


      PRE-CLINICAL TESTING


rBCG::RD1                             I Pasteur



rBCG DureC Hly                        MPIIB




Attenuated M. tuberculosis phoP       UNIZAR




LIVE VACCINE CANDIDATES FOR PHASE I
             Live Vaccines Based on Attenuated M. tuberculosis

              Which Genes to Choose for Inactivation?
                           (M. tuberculosisis genome near 4.000 genes)

       A PARTICULAR Mycobacterium bovis STRAIN CAUSED XDR OUTBREAK

                                                    First isolated in 1991

                                                   HIV+ individuals, 114 deaths

                                                    Highly transmissable by aerosol
                                                  route

 M. bovis B strain increased expresion of transcription factor gene phoP
                    THE PHOP/R TWO-COMPONENT SYSTEM OF MTB


Signal Transduction Systems enable bacteria to detect
environmental stimuli and respond to them
                                                            ATP
                   Sensor Protein
                                                            ADP                       P
               Transcription Factor
GGM
                   START POINT: INACTIVATION OF phoP
         INACTIVATION OF phoP IN A CLINICAL ISOLATE OF M. TUBERCULOSIS




M. tuberculosis                                             SO2 M. tuberculosis phoP-

                      SO2 phoP PROTOTYPE VACCINE



      Cording
                               M. tuberculosis                        Loss of Cording


                             phoP             phoR



                                                                     Attenuation in vitro
 Macrophages
 multiplication          SO2 M. tuberculosis phoP-

                             Km r                  phoR


  CFUs in lung,               Pérez et al. Mol Micro 2001
  liver & spleen                                                       Attenuation in vivo
                                               LIVE VACCINE TESTING
                                            TB Vaccine Cluster FP5 / TBVAC
      STEP-BY-STEP PRECLINICAL TESTING STRATEGY BASED ON Douglas Young “Road Map”


           1                                          2                                                             3

                                                          Safety                                       Immunogenicity
                                                                                                         Protection
      Construction
      Live                        Cell                               Animal Model                              Animal Model
      Vaccine




                                                                                     Guinea pigs (5)
                                                          Mice (1,2,4)




                                                                                                       Mice (3,4)


                                                                                                                        Guinea pigs
                                    Macrophages (1)




      Candidate




                                                                                                                                              Primate
                                                                          Mice (3)




                                                                                                                                      (2,3)
                                                                           SCID




                                                                                                                                                        (6)
 Preclinical Testing SO2 2001                                                                                                                                 2009
(1)                                                                                                                               (5)
       Mol Micro Perez et al 2001, (2) Tuberculosis Williams et al 2005, (3) Vaccine Martín et al 2006, (4) CEI Aguilar et al 2007(5)
(2)    Vaccine Cardona et al 2009 (6) Plos ONE Verreck et al 2009
                                            PhoP


                                   PHOP-REGULATED GENES

                TRANSCRIPTOMICS                     PROTEOMICS                  LIPIDOMICS
                McGill University                   ID Lelystad                 Institut Pasteur
                M. Behr                             J Thole                     B Gicquel




2% OF M. tuberculosis GENOME            SINGLE MUTATION PhoP      PhoP controls synthesis of
   UNDER PhoP CONTROL                      H37Rv /H37Ra:          sulfolipids,         and
                                                                  polyketides DAT and PAT.
                                                                  Chesne-Seck et al.
  Virulence genes: lipF…………..                                     J Bacteriol 2008
  SL, PAT, DAT , ESAT6

  74 mRNAs with decreased expression
  in MT103ΔphoP::hyg                                              PhoP     controls   ESAT-6
                                                                  secretion and specific T
  4 mRNAs with higher expression                                  cell recognition. Frigui et al.
  in MT103 DphoP::hyg                                             PLoS Pathog 2008
                                       I. FROM SO2 TO MTBVAC01
     CONCEPT, CONSTRUCTION AND PROOF OF PRINCIPLE OF phoP-BASED VACCINE


                ~2% ORFs in the M. tuberculosis genome under PhoP control

                                            RESPIRATION             STRESS
                                           narK1, nirA, cysH,       PROTEINS
    LIPID METABOLISM                       ald, nuoBCDK             (hsp) groEL2   RD1 REGION
     SL, DAT,PAT +.... lipF                                                        Secretion ESAT6

                                                        +




                                                                +
                                                    +   PhoP          +


                                                                -
                                                        +


                                          HYPOXIC                   icl
                                          RESPONSE

                                          INITIAL               PERSISTENCE
                                          dosS/R
                                                                                       Frigui et al. PLoS Path 2008
     Gonzalo Asensio et al. JBC 2006
                                          ENDURING
Gonzalo-Asensio et al.
PLoS ONE 2008
 gene name   Rv number   Z-Score
 bglS        Rv0186        2,17
 Rv0250c     Rv0250c       2,64
 hsp         Rv0251c       2,14
 groEL2
 icl
 fadB2
             Rv0440
             Rv0467
             Rv0468
                           2,32
                          -2,71
                          -2,82
                                    PhoP is a global regulador of intracellular
 umaA1       Rv0469       -2,10
 mmpS5
 phoP
 phoR
             Rv0677c
             Rv0757
             Rv0758
                           2,46
                           2,96
                           2,43
                                    style of live in M. tuberculosis
 phoY2       Rv0821c       2,25
 lpqS        Rv0847        2,65
 Rv0967      Rv0967        2,53
 Rv0968      Rv0968        2,19
 PE          Rv1040c      -2,70
 pks3        Rv1180        2,08
 Rv1184c     Rv1184c       3,12
 fadD21      Rv1185c       3,32
 Rv1217c     Rv1217c       2,14
 Rv1218c     Rv1218c       2,21
 Rv1219c     Rv1219c       2,33
 Rv1639c     Rv1639c       3,75
 Rv1687c     Rv1687c       2,64
 Rv1812c     Rv1812c       2,17
 Rv1986      Rv1986        2,51
 Rv1996      Rv1996        2,53
 Rv2137c     Rv2137c       2,61
 narK1       Rv2329c       2,82
 Rv2376c     Rv2376c       2,65
 Rv2389c     Rv2389c       2,47
 Rv2390c     Rv2390c       3,33
 nirA        Rv2391        5,14
 cysH        Rv2392        3,82
 Rv2393      Rv2393        3,14
 PE_PGRS
 fas
             Rv2396
             Rv2524c
                           4,08
                           2,12
                                          M. tuberculosis GENES IMPLICATED IN PERSISTEN INFECTIONS
 fadD9
 Rv2621c
             Rv2590
             Rv2621c
                           3,14
                           2,03    PhoP
 Rv2628      Rv2628        2,24
 Rv2630      Rv2630        2,08
 Rv2641      Rv2641        2,48
 Rv2642      Rv2642        2,14
 35kd_ag     Rv2744c       2,21
 ald         Rv2780        2,19
 Rv3127      Rv3127        2,51
 Rv3129      Rv3129        3,44
 dosS        Rv3132c       2,19
 dosR        Rv3133c       2,25
 PPE         Rv3135        4,08
 PPE         Rv3136        2,62
 Rv3137      Rv3137        2,74
 fadB4       Rv3141        2,11
 Rv3143      Rv3143        2,50
 nuoB        Rv3146        2,13
 nuoC        Rv3147        2,03
 nuoD        Rv3148       2,01
 nuoK        Rv3155       2,14
 Rv3161c     Rv3161c      2,05
 Rv3197      Rv3197       2,54
 Rv3269      Rv3269       2,18
 ctpC        Rv3270       2,36
 PE          Rv3477       3,20
 lipF        Rv3487c      3,17
 Rv3822      Rv3822       2,72
 pks2        Rv3825c      2,23
 Rv3849      Rv3849       3,36
 whiB6       Rv3862c      2,59
 Rv3864      Rv3864       2,13
 Rv3865      Rv3865       2,11
 Rv3866      Rv3866       2,11
 Rv3867      Rv3867       2,18
 PPE         Rv3873       2,37
 Rv3876      Rv3876       2,46
 Rv3877      Rv3877       2,40
 Rv3878      Rv3878       2,89
 Rv3879c     Rv3879c      2,62
 Rv3880c     Rv3880c      3,33


                                      Nature Reviews Microbiology Stewart, G.R., Robertson, B.D., & Young, D.B (2003)
 espB        Rv3881c      2,41

Gonzalo-Asensio et al. 2008
FUNDAMENTAL RESEARCH




    DEVELOPMENT
  GENEVA CONSENSUS CRITERIA: CONSTRUCTION OF MTBVAC01
   TWO STABLE INDEPENDENT MUTATIONS RECOMMENDED FOR LIVE VACCINES




DIM locus was found as a virulence gene cluster of M. tuberculosis by signature-tagged transposon
mutagenesis Camacho et al. Mol Microbiol 1999; Cox et al. Nature 1999

                                      MT103 fadD26-
                                      MT103           ATTENUATION and protection of M.
                                                      tuberculosis fadD26 mutant. Infante et al.
                                                      Clin Exp Immunol 2005




                   SECOND MUTATION IN DIM LOCUS: DELETION IN fadD26 GENE
                    FROM SO2 TO MTBVAC01
               STEP-BY-STEP CONSTRUCTION OF PHOP-/PDIM- VACCINE



    Mt103              phoP                       fadD26




     SO2                Km                        fadD26
                                                            1999 - 2001


     SO2
     ΔfadD26::Hyg       Km                       Ω Hyg
                                                            Feb 2006


    SO2
    ΔfadD26             Km
                                                            Oct 2006


     SO2
     ΔphoP::Hyg        Ω Hyg
     ΔfadD26                                                May 2007


                                  MTBVAC01
     SO2
     ΔphoP
     ΔfadD26                                                April 2008



GMP Good Manufacturing Practices, GCPs Good Clinical Practices            Ainhoa Arbués
Existing knowledge


     Products

Pre-clinical studies




    Industries ?

                       Clinical Research


                        New Vaccines
RESEARCH - DEVELOPMENT


      Product


 Pre-Clinical Studies




  INDUSTRIES

  OTRI UNIZAR
  GENOMA ESPAÑA


                         CLINICAL RESEARCH

                            NEW VACCINE
   MTBVAC01 TOWARDS PHASE 1



 2007              2008               2009               2010                  2011
         1Q   2Q     3Q   4Q   1Q   2Q    3Q   4Q   1Q    2Q 3Q     4Q    1Q   2Q 3Q 4Q




UNIZAR: Final
Construct  of
MTBVAC01      MTBVAC01
               Transfer
SO2
Transferred
to CMO

                                           Lyophilized Lot
                   Optimize
                                          of SO2 Prototype
                   Growth/Lyophiliz
                   ation of SO2

Clinical
Manufacturing             Final Construction of MTBVAC01: MEC-BIO2005-07949, EUTBVAC FP6
Organization
(CMO) Identified          TB-VAC PDT: G. Thiry, B. Walker, Mei Mei Ho, M. Roumiantzeff, Juhani Eskola
 MTBVAC01 TOWARDS PHASE 1



2007         2008                2009                2010                  2011
       1Q   2Q   3Q   4Q   1Q   2Q    3Q   4Q   1Q   2Q 3Q     4Q     1Q    2Q 3Q 4Q

                                     PDT/CDT/RAs - Additional Safety Studies


                                                                      Long Term Stability Data

                                                        Formal
                                                        Tox.   &
                                                        IND          Recruitment & Ph 1



                                                        Submission




                                GMP of MTBVAC01-
                                 Characterization
                                and QC Release of
                                   Final Product
                       Final Construction: MEC-BIO2005-07949, EUTBVAC FP6
                       TBVI PDT: (G. Thiry, M. Roumiantzeff and      B. Walker, Mei Mei Ho,
                       Juhani Eskola)
                          AIDS, MALARIA & TUBERCULOSIS




TUBERCULOSIS
Vaccines:

LIVE VACCINES                      TB cases per
                                   100 000



Protection Pulmonary TB
                                      300
                                      300
                                      or
  Replace BCG                         more


                     -                    Global Tuberculosis Control
                                          WHO Report
               GGM

http://genmico.unizar.es



Esther Perez
Henar Alonso                  Begoña Gracia
Isabel Millan                 Carmen Lafoz
José Antonio Aínsa*           Ainhoa Lucia
Ainhoa Arbués                 Isabel Otal*
Alberto Cebollada             Ana Pico
Nadia Lucía Ferrer            Sofía Samper *
Luis Solans                   Carlos Yesid Soto
Silvia Lobez
Ana Belén Gómez
Jesús Gonzalo-Asensio
Dessi Marinova



* IP coordinadores de línea   +   Microbiología Clínica + Medicina Preventiva
Institut Pasteur Paris (FR)
Brigitte Gicquel
                                               Lelystad (NL)
Mary Jackson
                                               Jelle Thole
                                               José Harders
IPBS CNRS Toulouse (FR)
Christophe Guilhot                                 Montreal (Canada)
                                                   Marcel Behr
                                                   Serge Mostowy
           Health Protection Agency (UK)
                                                          Biomedical Primate
           Ann Rawkins
                                                          Research Center (NL)
           Graham Hatch
                                                          Frank Verreck
           Simon Clark
                                                          Alan Thomas
           Philipp Marsh


         Universidad Autónoma                                  Unitat de Tuberculosi
         de México                                             Experimental HUGTP (ES)
         Rogelio Hernandez Pando                               Pere Joan Cardona
         Diana Aguilar                                         Olga Gil
                                                               Evelyne Guirado
                                                               Vicente Ausina
       Centro Nacional de Biotecnología (ES)
       Francisco García del Portillo

                                                      Universidad Autónoma
        University College Dublin (IRL)               de Madrid (ES)
        Eamonn Gormley                                María Jesús García
                                                      Carmen Menéndez
Artículo 130 de los Estatutos de la Universidad de Zaragoza 8. La Universidad velará para que sus
invenciones y contratos de investigación se utilicen para fomentar la paz y el desarme y para contribuir a la
desaparición de las desigualdades sociales y económicas entre las personas y entre los pueblos
Latest global TB estimates - 2007


                                        Estimated            Estimated
                                        number of new        number of
                                        cases                deaths

  All forms of TB                       9.27 million        1.77 million
  Greatest number of cases in Asia;
  greatest rates per capita in Africa
                                        (139 per 100,000)   (27 per 100,000)
  Multidrug-resistant
                                          511,000            ~150,000
  TB (MDR-TB)

  Extensively drug-
                                          ~50,000             ~30,000
  resistant TB (XDR-TB)

  HIV-associated TB                     1.4 million (15%)      456,000
Global TB Control Targets



2015: Goal 6: Combat HIV/AIDS, malaria and other diseases
   Target 8:       to have halted by 2015 and begun to reverse the
                   incidence…
   Indicator 23:   incidence, prevalence and deaths associated with TB
   Indicator 24:   proportion of TB cases detected
                   and cured under DOTS




2015: 50% reduction in TB prevalence and deaths by 2015
2050: elimination (<1 case per million population)
Sixty-Second World Health Assembly

22 May 2009


Resolution on prevention and control of MDR-TB
  and XDR-TB

URGES all Member States:

(1) to achieve universal access to diagnosis and treatment of
    MDR-TB and XDR-TB as part of the transition to universal
    health coverage ..................

(2) to enhance quality and coverage of DOTS..

(3) to use all possible financing mechanisms..

(4) to increase investment in operational research and
    research and development for new diagnostics, medicines
    and vaccines to prevent tuberculosis.
Tuberculosis Treatment


  Standard anti-TB treatment for drug sensitive strains
     2 months INH + RIF + (PZA) + EMB
     4 months INH + RIF


Tuberculosis Drug Resistance

  MDR-TB strains are resistant to both isoniazid and
 rifampicin with or without resistance to other drugs.
The high social value of vaccines contrasts with their relatively low
           economic value, compared to pharmaceuticals


           Social Value


                                   Vaccines
                   Social value




                                                       Pharmaceuticals




                                  Cost
                                              Economic Value
  Rappuoli, Miller & Falkow, The Intangible Value of Vaccination, Science, 2002

								
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