Horng-Yunn Dou( 杜鴻運 ) Division of Infectious Disease National Health Research Institute Immune Regulation and Vaccine Development : Tuberculosis as an exempt

Estimated numbers of tuberculosis cases by country in Adapted from WHO Global Report 2009 Estimated numbers of tuberculosis cases by country in Adapted from WHO Global Report 2009

Pathogenesis 5Pathogenesis 5 What are the likely outcomes following exposure to open TB? Dormant TB (90%) well no TB disease not infectious to others Active TB (10%) ill likely to die if untreated infectious Infection ( %) No infection (70-90%) Exposure to TB Activation of infection results in disease

Acid-fast staining

世界衛生組織目前 TB 防治首要任務為防止潛伏性肺結核之復發 目前首要研究方向以防止 目前首要研究方向以防止防止潛伏性肺結核1.Biomarker 2.New drug 3.Novel TB vaccine

台灣目前 TB 防治政策 Active TB 新感染病例 主動篩檢與治療 LatentTB reactivation 半數以上 TB 為復發病例 潛伏性感染的介入性防治與基礎研究 疾管署與地方衛生單位國家衛生研究院與各醫療研究單位 1.Yang CY, J Infect Dis. 2007Jul 15;196(2): Dou,HY,BMC Infectious Diseases Dec 22;8:170.

Nature Medicine 2007,13,

Host pathogen interaction

Factors effecting outcome of exposure to Mycobacterium tuberculosis

Adapt from PLOSpathogen 2008,4:e The main migrations events of MTB BeijingHaarlem EAI LAM

Netherlands 荷蘭 1600 Ming Dynasty Portuguese 葡萄牙 1600 Second world war 1945 Beijing Strain 北京株 Haarlem Strain 荷蘭株 LAM Strain 拉美株 EAI Strain 東非株 Austronesian 南島 Before yrs China

Pathogenesis - ability of organism to cause disease - route of transmission - stability of organism - infectious dose - concentration of organisms per volume - origin of agent - condition of host

Cycle of mycobacteria entering and exiting dormancy Microbiology and Molecular Biology Reviews,2008,72:

Risk Factors for the Development of TB Disease Infection with HIV Injection of illicit drugs TB infection within the last two years Chest x-rays suggestive of previous TB Diabetes mellitus Silicosis Prolonged corticosteroid therapy Immunosuppressive therapy Certain types of cancer Severe kidney disease Certain intestinal conditions

Mycobacteria–host-cell interaction at the macrophage level

A simple schematic of the outcomes of Mycobacterium tuberculosis infection at the level of the infected host cell – normally a macrophage. APMIS 2009,117:

Candidate antigen: 1.recognized by the CD4+ lymphocyte of Mtb-immune individuals. 2.stimulate Th1-type responses including IFN-γ secretion. 3.activate macrophage.

What is a Vaccine? Vaccines Preventive Prevent infectious diseases by inducing immune response e.g. Influenza, Diphtheria, Pertussis, Polio Therapeutic Treat diseasese.g. Cancer, HIV, multiple sclerosis(Copolymer 1, glatiramer accetate)

Known facts and knowledge gaps in vaccine developmen

A.Live vaccine Auxotropic muntant of M. tuberculosis. Re-engineered BCG. Other attenulated strains of mycobacteria---M.vaccae, M.microti. Non-mycobacterial live vectors---Vaccina virus, attenulated Salmonella strain. B.Non-living vaccine Subunit proteins DNA vaccine Novel TB vaccine

Phase 1: vaccine safety Small studies (n=40-50) Safety primary outcome – Injection site reactions (pain, erythema,swelling) – Systemic reactions (fever, anorexia, fatigue, headache, muscle ache, joint pain) Dose response – Often done with dose escalation with interval safety assessment Preliminary immunogenicity – Antibody response

Phase 2: immunogenicity Larger studies (n= ) Initial smaller studies to confirm optimal dose (dose ranging) Usually randomized, blinded, multicentered Immunogenicity primary outcome Late phase 2 studies can have expanded safety as primary outcome Lot consistency for at least one study

Phase 3: Efficacy There are 3 options for showing vaccine efficacy: – Clinical endpoint – Immune response endpoints, if accepted by regulator (e.g., Hib vaccines, Hepatitis B vaccines) – “Animal Rule” (FDA), if certain criteria are met Expanded safety Immunogenicity in subset

Adapted from Tuberculosis 2009 Nicole Ritz et al

Human Vaccines 2009,5:70-78 Genealogy of BCG vaccines

Should we need an additional boosting vaccine ? Data from TCDC Therapeutic vaccineBCGBooster subunit TB vaccine or rBCG?

I. Evaluation of the protection efficacy of BCG and rBCG against Taiwan local strains of Mycobacterium tuberculosis in mouse model( 國科會計畫 ) Immunization with BCG or rBCG Infect with local strain of MTB Evaluation of protection efficacy

Immunogenicity of recombinant BCG expressing Ag 85B-CFP 10 fusing protein and IL-12

Macrophage rBCG-Ag rBCG-IL-12 CD4 ￥ ￥ ＋ ＋ Ag+IL-12 IFN-r TB

CD4CD8

PBS BCGrBCG-1rBCG-2 CD44 + IFN-r + cell number ％ (in 10 5 cells) ＊、 rBCG -1 ~ pMV261 / Ag85B fusion CFP-10 rBCG ＊、 rBCG -2 ~ pMV261 / Ag85B fusion CFP-10 + pVV16 / hIL-12 rBCG Memory CD4 T lympocytes generated by rBCG vaccine in lung of the mice

PBS BCGrBCG-1 rBCG-2 CD44 + IFN-r + cell number ％ (in 10 5 cells) ＊、 rBCG -1 ~ pMV261 / Ag85B fusion CFP-10 rBCG ＊、 rBCG -2 ~ pMV261 / Ag85B fusion CFP-10 + pVV16 / hIL-12 rBCG Memory CD8 T lympocytes generated by rBCG vaccine in lung of the mice

38 Immunogenicity of rBCG expressing Ag85B-CFP10 fusing protein and IL12

39 Antibody response against Ag85b and CFP10 in mice immunized with PBS, BCG, rBCG1 or rBCG2.

40 rBCG vaccination induced MCP-1, MIP-1α and Rantes expression in mice lung

Ex vivo Mycobacterial growth inhibition assay Peptide-specific CD4 + and CD8 + T cell response in the lung of vaccination mice

II.Development of Recombinant BCG or Subunit protein TB vaccine for latency ( 國衛院計畫 ) 1.rBCG :Ag85B+Latency antigen (RV2031c)+ESX gene(Rv2615c) 2. Subunit lipoprotein TB vaccine: Ag85B+ESPc

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