Transcript Correlates Minutes

Update on Immunoassays
& Correlates of Protection
1
Content
• Immunoassays to monitor vaccination status
in humans, NHP and Fischer 344 rats
• Functional assay to measure inhibition of
SCHU S4 growth in human PBMC as a
correlate of protection
2
LVS Vaccination Induces Humoral Immunity in
Humans and Fischer 344 Rats
Human
4
3
2
0.4
Unvaccinated
LVS vaccinated
0.3
OD405 nm
Unvaccinated 1
Unvaccinated 2
Unvaccinated 3
Vaccinated 1
Vaccinated 2
5
0.2
0.1
1
0
1:50000
1:25000
1:16000
Dilution
100000
1:8000
10000
1:5000
1000
1:2500
100
1:1600
0.0
1:800
OD405nm
Fischer 344 rats
3
IgG anti-LVS Units (Mean +/- S.D.)
S.C.
20000
2000
200
2
A06675
A06702
A07395
2E-1
A07418
20
A07566
2E-2
A07610
2E-3
Day 0
Day 7
Day 14
Day 21
Day Post-LVS Vaccination
Day 28
IgG anti-LVS Units (Mean +/- S.D.)
LVS Vaccination Induces Humoral Immunity in
NHPs Detectable by ELISA
Scarification
20000
2000
200
20
A06674
A06693
2
A06873
2E-1
A07386
A07682
2E-2
A07686
2E-3
Day 0
Day 7
Day 14
Day 21
Day 28
Day Post-LVS Vaccination
4
LVS Vaccinated NHPs are not Uniformly Protected
from Death due to SCHU S4 Aerosol Challenge
Despite Sero-conversion
SCHU S4 CFU Challenge
5000
Control
Scarification
S.C.
4000
3000
2000
1000
0
-1000
4
6
8
10
12
14
16
18
20
22
Days Survival Post-SCHU S4 Challenge
Day 21 = Scheduled euthanasia
5
LVS Vaccination Followed by SCHU S4 Challenge
Induces Ft-specific IFNg ELISpot responses in the
tracheobronchial lymph nodes
LVS vacc/ Schu S4
chall day 21
NHPs vaccinated with 3x105 LVS via scarification and challenged with ~750 SchuS4 via
bronchoscope instillation (approximately 6 months after vaccination)
6
Intracellular staining (ICS) of NHP cytokines by
flow cytometry
• Multifunctional T cells producing IFN-g, IL-2 and TNF-a
proposed to be important for control of some human
infections
• Goal: develop and optimize ICS for detection of
multifunctional CD4 and CD8 T cells in PBMC using 6 color
FACS Canto
• Flow cytometer settings have been optimized
• Currently optimizing timing post vaccination (> 6 d) and
duration of antigen-specific in vitro restimulation with hkLVS
and ffLVS (> 6h).
7
Planned Experiment
• Micro-agglutination assay – assays Francisella specific
antibodies in serum, binding to a standardized stained
particulate F.tularensis antigen
• This assay will bridge the animal models to earlier human
studies as this assay was traditionally used for clinical
human samples
8
Functional Assay to Determine
Protection Status
9
Assumptions Used to Develop Assays
for Protection Status
Unvaccinated
monocytes
T cell
LVS vaccinated
monocytes
T cell
T cell
Ft growth NOT
controlled
IFNg
Ft growth
controlled
TNF
10
Vaccinated Human PBMC Produce IFNg after
Antigen-specific Restimulation with Ft
250
Naive
Vaccinated
Spots/well
200
150
100
50
0
HK-LVS
FF-LVS
11
Experimental Design & Flow
1.
2.
monocytes
T cell
3.
monocytes
T cell
(2.5 x 106 c)
rIFNg ± rTNF
(100 U/ml)
monocytes
T cell
FF/HK-LVS
Prestim
(MOI= 1; 0-2 d)
Expected results
(MOI =1)
Unvacc – growth
Vacc– NO growth
Unvacc – growth
Vacc - growth
Complete
w/o cytokine – growth
w/ cytokine – No growth
Unvacc – growth
Vacc – NO growth
Complete
12
SCHU S4 Grows in Unvaccinated PBMC
1.010 6
CFU / well
1.010 5
1.010 4
1.010 3
1.010 2
MOI=1.0 (-) serum
MOI=1.0 (+) serum
1.010 1
1.010 0
0
24
48
72
hours post-infection
13
IFNg/TNF Activates Unvaccinated PBMC to
Control SCHU S4 Growth
IFN/TNF
IFN/TNF
Infect
IFN/TNF
-48 h
-24 h
0h
IFN/TNF
IFN/TNF
Harvest
24 h
48 h
72 h
total CFU / well (Log 10)
6
5
4
PBMC
+ IFNg
+ TNF
+ IFNg + TNF
3
2
1
0
3
48
72
Time post-infection (hours)
14
48 h Preactivation Sufficient to Control
SCHU S4 Growth in Unvaccinated PBMC
IFN/TNF
-24 h
total CFU / well (log 10)
-48 h
Infect
0h
Harvest
24 h
48 h
7
6
5
4
3
2
1
0
72 h
PBMCs:control
+ IFNg + TNFa
0
72
Hours post-infection
15
FF-LVS Stimulates Vaccinated Human PBMC
to Produce IFNg
LVS Vaccinated
Unvaccinated
30000
30000
Untreated
20000
pg/mL
pg/mL
IFNg + TNFa
FF-LVS
20000
10000
10000
0
0
-24
0
24
48
Time post infection (h)
72
-24
0
24
48
72
Time post infection (h)
16
Pre-stimulated Vaccinated PBMC Reduces
SCHU S4 Burden
Vaccinated
Unvaccinated
7
7
Vacc PBMCs:unstim
+ HK-LVS
+ FF-LVS
+ IFNg + TNFa
10)
6
total CFU / well (Log
Total CFU / well (Log 10)
6
Unvacc PBMCs
+ HK-LVS
+ FF-LVS
+ IFNg + TNFa
5
4
3
5
4
3
2
0
72
Hours post-infection
2
0
72
Hours post-infection
17
Currently Accumulated Results from
Human PBMC Studies
Absolute 72h CFU-fold increase
with FF-LVS stimulation
2500
-fold increase
2000
1500
1000
500
0
Vaccinated
Unvaccinated
18
Planned Experiments
• Continue screen of human vaccinees
– Vaccinated vs. unvaccinated
– Pre vs. post vaccination
• Develop functional assay in NHP and Fischer 344 rats
19
Summary
• Antibody ELISA and IFNg ELISpot assays have been
developed and optimized to monitor the vaccination
status of humans and animals.
– Development of multifunctional T cells staining and microagglutination assays are in progress
• Inhibition of SCHU S4 growth by human PBMC may
be potential functional correlates of protection
– Similar assays will be developed for NHP and rats
20