Immune Regulation and Vaccine Development :
Tuberculosis as an exempt
Horng-Yunn Dou(杜鴻運)
Division of Infectious Disease
National Health Research Institute
Estimated numbers of tuberculosis cases by
country in 2007.
Adapted from WHO Global Report 2009
Adapted from WHO Global Report 2009
t
h
o
g
e
n
e
s
i
s
5
What are the likely outcomes following exposure to open TB?
Exposure to TB
Infection
(10-30%)
No
infection
(70-90%)
Dormant TB (90%)
• well
• no TB disease
• not infectious to others
Active TB (10%)
• ill
• likely to die if untreated
• infectious
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
疾管署與地方衛生單位
主動篩檢與治療
國家衛生研究院與各醫療研究單位
潛伏性感染的介入性防治與基礎研究
新感染病例
半數以上
TB為復發病例
Active
TB
LatentTB
reactivation
1. Yang CY, J Infect Dis. 2007Jul 15;196(2):281-8.
2. Dou,HY,BMC Infectious Diseases. 2008 Dec
Nature Medicine 2007,13,284-285
Host pathogen interaction
Host factor:
Genetic resistance, nutritional
status(Iron, Vit D), Vaccination status
Environmental factor:
Immunomodulatory
influences, co-infection
Pathogen factor:
Mtb virulence
Factors effecting outcome of exposure to Mycobacterium tuberculosis
The main migrations events of MTB
Haarlem
Beijing
LAM
EAI
Adapt from PLOSpathogen 2008,4:e1000160
LAM Strain
拉美株
Beijing Strain
北京株
Portuguese
葡萄牙
1600
China
Second world war
1945
Ming Dynasty
1368-1644
Haarlem Strain
荷蘭株
EAI Strain
東非株
Netherlands
荷蘭
1600
Austronesian 南島
Before 5000-2500 yrs
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,2008,72:126-56.
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 2009,117:440-457
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
Novel TB vaccine
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
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=100-2000)
• 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
Genealogy of BCG vaccines
Human Vaccines 2009,5:70-78
Should we need an additional boosting vaccine ?
350
2005
2006
2007
2008
2009
2010
Rate ( per 100K)
300
250
200
150
100
50
0
0-4
5-9 10-14 15-19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 ≧65
Age group (years)
BCG Booster subunit TB vaccine or rBCG?
Data from TCDC
Therapeutic vaccine
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
Ag+IL-12
＋
CD4
rBCG-Ag
Macrophage
TB
CD4
rBCG-IL-12
＋
IFN-r
Challenge vaccine after 4 weeks
Challenge vaccine after 4 weeks
450
Number of cells / 104 cells
CD4
CD8
250
200
150
100
50
Number of cells / 104 cells
300
0
IL-2
IFN-γ
TNF-α
400
350
300
250
200
150
100
50
0
＋
－
－
－
＋
－
－
－
＋
＋
＋
－
＋
－
＋
－
＋
＋
＋
＋
＋
IL-2
IFN-γ
TNF-α
＋
－
－
Challenge vaccine after 8 weeks
Number of cells / 104 cells
Number of cells / 104 cells
200
150
100
50
0
＋
－
－
－
＋
－
－
－
＋
＋
＋
－
＋
－
－
＋
＋
＋
Challenge vaccine after 12 weeks
＋
＋
＋
＋
－
＋
－
＋
＋
＋
＋
＋
－
＋
＋
＋
＋
＋
400
350
300
250
200
150
100
50
0
IL-2
IFN-γ
TNF-α
＋
－
－
－
＋
－
－
－
＋
＋
＋
－
＋
－
＋
Challenge vaccine after 12 weeks
300
Number of cells / 104 cells
300
Number of cells / 104 cells
＋
＋
－
Challenge vaccine after 8 weeks
250
250
200
150
100
50
0
IL-2
IFN-γ
TNF-α
－
－
＋
450
300
IL-12
IFN-γ
TNF-α
－
＋
－
250
200
150
100
50
0
＋
－
－
－
＋
－
－
－
＋
＋
＋
－
＋
－
＋
－
＋
＋
＋
＋
＋
IL-2
IFN-γ
TNF-α
＋
－
－
－
＋
－
－
－
＋
＋
＋
－
＋
－
＋
－
＋
＋
＋
＋
＋
CD44+ IFN-r+ cell number ％
(in 105 cells)
Memory CD4 T lympocytes generated by rBCG vaccine in lung of the mice
30
25
20
15
10
5
0
PBS
BCG
rBCG-1
rBCG-2
＊、rBCG -1 ~ pMV261 / Ag85B fusion CFP-10 rBCG
＊、rBCG -2 ~ pMV261 / Ag85B fusion CFP-10 + pVV16 / hIL-12 rBCG
CD44+ IFN-r+ cell number ％
(in 105 cells)
Memory CD8 T lympocytes generated by rBCG vaccine in lung of the mice
30
25
20
15
10
5
0
PBS
BCG
rBCG-1
rBCG-2
＊、rBCG -1 ~ pMV261 / Ag85B fusion CFP-10 rBCG
＊、rBCG -2 ~ pMV261 / Ag85B fusion CFP-10 + pVV16 / hIL-12 rBCG
Immunogenicity of rBCG expressing Ag85B-CFP10
fusing protein and IL12
Table 1. BCG and rBCG vaccination increased non-specific production of TNF-α
No stimulus
M.tuberculosis H37Rv
P.aeruginosa
PBS
BCG
0
0
0
0
16.78±10.66
23.53±4.94
rBCG1
0
66.82±25.28
73.18±8.63
rBCG2
0
168.09±15.13
237.83±13.61
38
Antibody response against Ag85b and CFP10 in mice immunized with PBS,
BCG, rBCG1 or rBCG2.
39
rBCG vaccination induced MCP-1, MIP-1α and Rantes expression in
mice lung
40
Peptide-specific CD4+and CD8+ T cell
response in the lung of vaccination mice
Ex vivo Mycobacterial growth inhibition assay
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
Nature Medicine2011 SHE Kaufmann