advances in tb vaccinology
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Advances in TB vaccinology. There have been several major changes in the way we look at vaccine in development in the last few years: Advances in bioinformatics/genomics at the bacterial level Identification of targets for vaccines has expanded enormously - PowerPoint PPT PresentationTRANSCRIPT
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Advances in TB vaccinology
There have been several major changes in the way we look at vaccine in development in the last few years:
1. Advances in bioinformatics/genomics at the bacterial level
a) Identification of targets for vaccines has expanded enormouslyb) We are beginning to understand how M. tuberculosis reacts to the host’s
immune response
2. Advances in understanding immunology and disease processes in patients and animal models
a) Choosing targets and designing interventions has become much more sophisticated
b) Our understanding of what constitutes a desirable immune response has broadened
3. The first clinical trials have started
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Antigen discovery in the pre-genomic era
2244
2244
3765
The Ag85 family(Ag85A,B and C)
The ESAT6 family(20 members
organized pairwise) +
+
+
+ +++
+ +++ +
+ + +
+
++
+
++
+
++
+
+
+ + + +
++
++ + +
+ +
++
++
++
+++
+ + ++
+++++
++ +
+++
+++ ++
028802880288
38753875
3648c
02871038c/1197/1792
39142031c
2031c
2878c1926c0652
3874
3418c
2140c 0009 0009 0009
1984c
1932
1827
2534c
+2882c
3803c2109c
2109c1980c
07333036c
05771886c 0129c 3804c
0363c
0798c271616260036c
09343045
27802780278027800884c
04621098c
18601860
18601860
1860
2220
1077
3842c
0350
Definition of secreted antigens
Size fractionation
Antigen recognition
14 21 31 45 66 97
Mouse(93) Cattle(97)
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Human recognition of antigens(ranked by mean value of responses)
Ag85B
ESAT 6
CFP 10
TB 10.
4
TB 9.5
6
TB 37.
6
TB 12.
3 pep
.
TB 9.5
8
TB42.9
TB 10.
3
TB 27.
4 pep
.
TB 7.7
pep
.
TB 9.8
1
TB 12.
9
0
25
50
75
100100
200
300
400
%re
spo
nse
of
PP
D
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Finding Vaccine Targets
Of the hundreds of antigens screened, the vast majority are not strongly immunogenic.
However, of the the antigens that are immunogenic, most come from a relatively small number of gene families.
Thus, looking at genomic organization has proven to be a very efficient route of finding antigens to screen.
Many of these antigens have also proven to be virulence factors, suggesting that functional analysis might also be a useful way to identify targets
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Lifetime risk of Tuberculosis (the clinician’s view)
Exposure
Healthy (97%)
Year 1 Year 2 Year 3 thereafter
TB (3%)
Healthy (95%)
TB (2%)
Healthy (94%)
TB (1%)
Healthy (approx. 90%)
TB (less than 0.1%/year)
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Acute infection Latent infection
Expression of early phase Expression of late phase genesgenes such as Ag85 such as a-crystallin and and ESAT-6 the DosR regulon
Immune response initiated Immune response alters
Progress of infection (the microbiologist’s view)
CF
U
Acute Disease
Reactivation of infection
Years after exposure
1-3 4-50
Elimination?
Latent infection
Immune conversion
Latency?
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Alteration of antigen recognition as disease progresses
10
100
1000
10000
TB HHC LTBI
p<0.001
p<0.001
Rv2031c response in clinical groups
10
100
1000
10000
TB HHC LTBI
ESAT-6 response in clinical groups
IFN
- (
pg/m
l)
TB HHC LTBI0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Clinical status
Lin
ear
regr
essi
on o
f ra
tio
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Response to infection (the immunologist’s view)
Early bacterial growth arrested at early time point. May (or may not) result in latent infection
Initial infection
Early bacterial growth not contained. Leads to clinical illness
Subsequent bacterial growth contained. Symptoms abate but latent infection established.
Bacterial growth not contained. Progressive and eventually fatal disease unless treated
Reactivation of latent infection at a later point in life
33%
67%
8%
25%
2%
Remain healthy but latently infected
23%
These individuals do not apparently skin-test convert or become ESAT-6 positive
These individuals generally skin-test convert and become ESAT-6 positive. They often have characteristic patterns on X-ray.
Immunologically these individuals tend to express elevated levels of IL-4 and in advanced disease, decreased IFN- and IL-12
Immunologically, these individuals tend to express elevated levels of IFN- and IL-12, and while IL-4 often remains slightly increased, its antagonist IL-42 is greatly increased
Immunologically, little is known about these individuals as they cannot be distinguished from uninfected individuals
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The balance of Th1 and Th2 cytokines - not the absolute level - correlates with disease status
Rat
io I
L-4
/IL-
42
Rat
io I
L-4
/IF
N-
TB HHC LTBI0.0
2.5
5.0
Clinical status
TB HHC LTBI0
5
10
15
p<0.001
p<0.01
p<0.001
p<0.01
mR
NA
exp
ress
ion
in u
nstim
ulat
ed P
BM
C
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The infection process (the cell biologist’s point of view)
PAMP binding
IL-12
IL-12R
IFN- IFN-R
Uptake/Phagocytosis
Lysosome maturation and bacterial killing
Jak/Stat activation
TNF-
TNF-R
Presented antigen
Specific T cell proliferation
MHC II
T cell Antigen presenting cell
M. tuberculosis
Stat1 activation
IL-18IL-18R
Mycolic acids, lipoproteins
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The infection process (the cell biologist’s point of view)
PAMP binding
IL-12
IL-12R
IFN- IFN-R
Uptake/Phagocytosis
Lysosome maturation and bacterial killing
Jak/Stat activation
TNF-
TNF-R
Presented antigen
Specific T cell proliferation
MHC II
T cell Antigen presenting cell
M. tuberculosis
Stat1 activation
DC-SIGNIL-10
IL-10R
IL-10R
IL-18IL-18R
PGL Mycolic acids, lipoproteins
LAM
Multiple factors
ESAT-6/ CFP10
Bacterial lipid-induced IL-4/13
Decoy antigens (27 kDa, PE/PPE family)
19 kDa
LAM
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What does this mean from a vaccine designer’s perspective?
The fact that most exposed individuals develop a protective immune response, shows that immunity is possible
The fact that even taking this into account, BCG can reduce mortality shows that boosting that immune response is possible
The fact that BCG has not performed well against adult pulmonary TB shows the need for a new vaccine
The fact that M. tuberculosis can survive for extended periods in people with strong antigen-specific immune responses, shows that a strong IFN- response is not, by itself, enough
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Why can’t BCG be used against adult pulmonary TB?
BCG
Systemic protection
BCG
BCG Eliminated
No protection
Proliferation and
dissemination
Naive Recipient
Sensitized Recipient
Pre-existing immunity
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BCG - boost or replace?
Booster vaccine
BC
G-i
nduc
ed le
vel o
f im
mun
ity
0-14
15-24
25-34
35-44
45-54
55-64
65+
Age (years)
Improved priming vaccine
BC
G-i
nduc
ed le
vel o
f im
mun
ity
0-14
15-24
25-34
35-44
45-54
55-64
65+
Age (years)
After Hart, 1977 and Sterne, 1998
TB
inci
denc
e pe
r 10
0,00
0
0-14
15-24
25-34
35-44
45-54
55-64
65+
Age (years)
0
250
500
750
1000
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Vaccines in, or on their way to, clinical trials
Vaccine Name Notes
rBCG30 Live, recombinant BCG, over-expressing Ag85B from M. tuberculosis.
Currently in clinical phase I trials.
rBCG:: D ureC-
llo+
Live, recombinant BCG, urease-deficient mutant which expresses the
Lysteriolysin O ge ne from Listeria monocytogenes. Currently scheduled to
enter clinical trials in 2005/2006.
MVA-85A Live, recombinant, replication deficient vaccinia virus, expressing Ag 85A
from M. tuberculosis. Currently in clinical trials.
Ag85B-ESAT6 Recombinant protein, composed of a fusion of ESAT-6 and Ag85B
from M. tuberculosis. De livered in the IC31 adjuvant or i n cationic
liposomes. Clinical trials planned in 2005.
Mtb72f Recombinant protein, composed of a fusion of Rv1196 and Rv0125
from M. tuberculosis. Delivered in an oil-in-water emulsion containing
the immunostimulant 3-deacylated-monophosphoryl lipid A and a
purified fraction of Quillaria saponaria, (Quil A). Currently in clinical
phase I trials.
Prim
ing
vacc
ines
Boo
stin
g va
ccin
es
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Priming vaccines (BCG-derived)
rBCG30
• Recombinant BCG Tice which over-expresses Ag85b
• Protects guinea pigs better than BCG
• Completed phase I trials - vaccine is immunogenic and apparently safe - currently being reworked for further testing
rBCG::ureC-llo+
• Recombinant BCG which expresses Lysteriolysin O to cause leakage from the endosome, and and urease C to alter vacuole pH. The idea is to improve CD8 response via “cross-priming”
• Protects mice better than BCG, but is less virulent than BCG
• Clinical trials planned for 2006/7
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Priming vaccines (M. tuberculosis-derived)
PanD-/Leu- auxotroph
• Recombinant M. tuberculosis lacking both the PanD and Leu genes
• Protects guinea pigs and is much less virulent than BCG: but it also grows less well in the host, so is slightly less protective
phoP/R
• Recombinant M. tuberculosis in which the phoP virulence factor has been knocked out by the insertion of an antibiotic gene
• Protects guinea pigs better than BCG and is less virulent
• Will probably need further manipulation before it could be used in human trials
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Priming vaccines (viral vectors)
MVA85A
• Recombinant, replication deficient vaccinia virus, expressing the strongly immunogenic antigen 85A from M. tuberculosis
• Protects mice and guinea pigs as well as BCG, can boost BCG effect
• Has completed early clinical trials: is apparently safe and immunogenic
Other viruses
• Adenovirus - a variety of different constructs have shown efficacy in animal models
• Fowlpox - has also shown efficacy in animal models
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Priming vaccines (recombinant proteins)
ESAT-6-Ag85B
• Recombinant fusion protein, composed of the strongly immunogenic antigens ESAT-6 and Antigen 85B from M. tuberculosis
• Two forms of the vaccine using different adjuvants
• IC31, for intramuscular administration
• LTK63 for nasal administration
• Protects mice and guinea pigs as well as BCG, can boost BCG effect
• Will enter clinical trials in September 2005
72f
• Recombinant fusion protein, composed of the strongly immunogenic antigens Rv1196 and Rv0125 from M. tuberculosis
• Administered in AS2 adjuvant
• Protects mice and guinea pigs as well as BCG
• Has completed early clinical trials: is apparently safe
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Adjuvants for human use
Recombinant vaccines are now routinely used in humans, but only for limited categories of disease - there are few adjuvants that combine low toxicity with the ability to stimulate good CMI responses
However, our improving understanding of the interaction of bacteria and the immune system - primarily through APCs - has led to the development of new adjuvant systems that mimic bacterial infection and which look promising for new vaccines.
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Adjuvants for human use
Already approved for human use
Alum (AlOH)
MF59
Virosomes
The first human adjuvant, alum promotes a strong humoral response and is widely used in viral vaccines. However it generates strongly Th2-polarised responses and is not suitable for use as a TB vaccine.
An oil-in-water emulsion composed of 5% v/v squalene, 0.5% v/v Tween 80 and 0.5% v/v Span 85. Like alum, it generates primarily humoral immunity and is currently used mostly in influenza vaccines
Similar in structure to liposomes, virosomes are differentiated by containing viral proteins embedded in their membrane, which are delivered into host cells by membrane fusion. Currently used in vaccines against viral targets such as influenza and hepatitis A, where humoral immunity is most important.
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Adjuvants for human use
Adjuvants tested in clinical trials
CAP (calcium phosphate) nanoparticles. Currently in early clinical trials, CAP have been used to generate humoral responses, but the lower levels of IgE induced suggest it may not be as polarized towards the Th2 pole of the immune response as alum.
LTK63. A modified and detoxified heat labile toxin from Escherichia coli tested in human volunteers as an influenza vaccine. Generates strongly Th1-polarised responses and therefore being considered for vaccines against M. tuberculosis and HIV.
AS2. An oil-in-water emulsion containing 3-deacylated-monophosphoryl lipid A(a detoxified form of lipid A from Salmonella minnesota), and a purified fraction of Quillaria saponaria, known as Quil A. Currently in early clinical trials as a TB vaccine. A synthetic analogue of monophosphoryl lipid A called RC-529) is in clinical trials in an HIV vaccine. Generates strongly Th1-polarised responses.
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Adjuvants for human use
Adjuvants in, or approaching, clinical trials
IC31. A mixture of oligodeoxynucleotides and polycationic amino acids. Generates strong Th1 responses and planned to enter phase I clinical trials in 2005 as part of a TB vaccine.
Montanide. A water/oil emulsion, two variants exist, based on mineral and non-mineral oil. Tested initially as a cancer immunotherapeutic agent, Montanide has now been through a variety of clinical trials through to phase III. It generates a mixed cell-mediated and humoral response, which may render it less attractive for a TB vaccine.
ISCOM. A formulation of Quillaja saponins, cholesterol, phospholipids, and protein, typically self-assembling into small icosahedral cage-like particles. Used initially for veterinary vaccines, ISCOMS have recently shown promise in late phase human clinical trials for viral vaccines. Their potential for M. tuberculosis vaccines remains unknown, as they generate a mixed humoral and cell-mediated response.
OM-174. A modified and detoxified lipid A from Escherichia coli. Synthetic analogues also exist. Currently in early clinical trials for cancer immunotherapy and suggested for TB vaccine use. Generates strongly Th1-polarised responses .
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Shared characteristics for ”good” TB adjuvants
Cationic vehicle – interacts with cell membranes and accelerates antigen uptake
Immunomodulator – activates APC/DC
Cationic Liposomes (CAT-1/2)• DDA• MPL-A (Monophosphoryl lipid)
or• TDB (synthetic cord factor)
IC31• Poly leucine/lycine peptide (KLKLLLLLKLK)• Poly-IC analogue (TLR 3/9)
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Shared characteristics for ”good” TB adjuvants
Vehicle – forms a depot, for longer release
Immunomodulator – activates APC/DC
LTK63• a modified, heat-labile enterotoxin from E. coli
AS2• A fraction of Quillaria saponaria, known as Quil A.• 3-deacylated-monophosphoryl
lipid A
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are needed to see this picture.
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New TB Vaccines Today
• There are 5 novel vaccines in the early clinical pathway
• There are at least as many vaccines in the preclinical phases
• We have novel delivery systems that can be used for TB vaccines
• However….• None of these vaccines have yet shown proof of efficacy in humans• It is unknown if these vaccines will be effective in people who are already infected
• Research on improving TB vaccination is therefore still very much ongoing
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TB research at the Dept. of Infectious Disease Immunology, SSI:
ImmunologyAnja OlsenElse Marie AggerSøren HoffThomas BennekovKaren KorsholmMark DohertyJes DietrichCarina V. LundbergClaire AndersenJesper Davidsen
Protein chem.Ida RosenkrandsKarin Weldingh
Molecular biologyClaus Aagaard
Head.Peter Andersen