Progress Report
11/4/2009
TVDC team – UNM
Prepared by Terry Wu
1
Active Milestones
10. Testing vaccine candidates:
Aduro’s Lm-based vaccines in mice and rats
11. Cellular and humoral immunity in LVS vaccinated Fischer rats:
Role of T cells in passive immunization
12/13. Immunoassays (discussed at the LBERI tech call)
Correlation between CFU and protein content
Micro-agglutination assay
21. Correlate of protection assay using human PBMC
New data from 3 prevaccination samples, 1 control, 1 LVS vaccinated
samples
29. Result from screening individual polypeptides
PPG. Protection of NHP by vaccination with LVS lot 16
2
MS 11: Characterization of Fischer 344 Rat
Fischer 344
rats
Humoral
immunity
Cell mediated
immunity.
LVS vaccination
Purchase and culture hybridoma
cell lines
Passive transfer of
serum
Production of ascites fluid for CD4 and
CD8 depletion
Protection against i.t
SCHU challenge
In vivo depletion
Adoptive transfer
Characterizing elements
of protection
Blue: Steps in the milestone
Red: Completed
Green: In progress
Protection against i.t. SCHU
challenge
3
Milestone 11: Humoral Immunity
• Antibody responses develop within 7 days of s.c. LVS
vaccination; IgG2a, IgG2b > IgG1, IgG2c, IgM, no IgA
• Passive immunization with immune serum or purified IgG
protected rats against low dose i.t. SCHU S4 challenge
• Intermediate phenotype between NRS and LVS vaccinated rats:
bacteriology & histopathology, disease severity
• No increase in protection with larger antibody dose or with
repeated antibody treatment
• Completing experiments for manuscript
4
Is Passive Immunity Dependent on T cells?
• T cell depletion prior to serum treatment and challenge
• Nude rats
1)
2)
3)
4)
anti-CD4
anti-CD8
anti-CD4/8
Isotype ctrl
-2
i.t. infection
(~250 SCHU S4)
-1
0h
Survival
+
Weekly antibody treatment
30
1) Normal serum
2) Immune serum
5
Percent survival
Protection Requires CD8 T cells and
Partially CD4 T cells (Expt 1)
100
Depletion / treatment
80
None / NRS
Isotype Ctrl / IRS
CD4 / IRS
CD8 / IRS
CD4+CD8 / IRS
60
40
20
0
0
5
10
15
20
25
30
Days Post-challenge
Challenge dose 200 CFU/rat
6
Percent survival
Protection Requires CD8 T cells but not
CD4 T cells (Expt 2)
100
Depletion / Treatment
80
None / NRS
Isotype ctrl / IRS
CD4 / IRS
CD8 / IRS
CD4+CD8 / IRS
60
40
20
0
0
5
10
15
Days Post-challenge
Challenge dose 150 CFU/rat
7
Extended Survival in Nude Rats?
RNU rats:
Mixed genetic background
no T cells, but have B cells and higher numbers of NK cells
Percent survival
100
75
Normal serum (n = 2)
Immune serum (n = 3)
50
25
0
0
5
10
15
20
Days
Challenge dose 150 CFU/rat
8
Milestone 11 Humoral Immunity: Plans
• Confirm CD4 inactivation by functional assay
• Continue manuscript preparation
9
Milestone 11 Cellular Immunity: Plans
• Experiment in progress to determine the
requirement for T cells in LVS vaccinated rats
• Develop method for adoptive transfer of immune T
cells into naïve rats (technique reported for Fischer
344 rats)
10
MS 21: Assays in Vaccinated Humans
Assay to measure activation of PMBC killing
mechanisms in humans
Determine the approximate
yield of PBMC from whole blood
(1-200 ml max)
Determine and optimize cell
number and MOI
Evaluate assay with
IFNg and TNF
Develop assay with F. tualrensis
Compare human vaccinees and controls
Blue: Steps in the milestone
Red: Completed
Green: In progress
Statistical analysis
11
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
MS21: Background
• Human PBMC supports SCHU S4 growth
• Pre-stimulation of vaccinated human PBMC with
formalin-fixed LVS
– induced IFNg production within 24 h
– inhibited /reduced SCHU S4 growth @ 72 h post infection
Wash
SCHU S4
FF LVS
-48 h
-24 h
0h
Burden
24 h
48 h
72 h
• Activities correlates with vaccination status
13
Accumulated Results from
Human PBMC Studies as of Sept 2009
Absolute 72h CFU-fold increase
with FF-LVS stimulation
2500
-fold increase
2000
1500
1000
500
0
Vaccinated
Unvaccinated
Currently Accumulated Results from
Human PBMC Studies – Oct 2009
CFU-fold increase with 48-hour
FF-LVS pretreatment, absolute
4000
FT-AH-#32
(UBS)
-fold increase
3000
#31(prevacc)
2000
1000
#28
#29(prevacc)
#30(prevacc)
0
vaccinated
unvaccinated
6
Planned Experiments
• Continue screen of human vaccinees
– Vaccinated vs. unvaccinated
– Pre vs. post vaccination
– Meet with statistician
• Determine the mechanism of inhibition, e.g.
inhibiting with anti-IFNg antibody
• Develop functional assay in NHP and Fischer 344 rats
MS 10: Testing Vaccines Candidates
Cerus/Aduro
ASU
UTSA
Others
UNM
BALB/c
mice
Blue: Steps in the milestone
Red: Completed
Green: In progress
Fischer 344
rats
17
Milestone 10: In progress
•
•
•
•
Candidates selection based on protection against LVS in mice
BH2172 (Live Lm677 KatG-SL8)
BH2182 (Live Lm677 IglC-B8R)
Mice
6 wk
Prime (i.v.)
boost (i.v.) 4 wk challenge (i.v. & i.n.)
• Rats
Prime (i.v.) 6 wk boost (i.m.)
4 wk
challenge (i.t.)
4 wk
boost (i.m.)
18
Others
• MS 12/13: Immunoassays
– Microagglutination
• Stain LVS lot 9 with hematoxylin
• Develop assays with sera from human, rat, mouse
– Normalization of antigen preps
• MS 29: Identification of antigenic peptides in
ASU peptide library
– Waiting for protection results from LVS lot
comparisons
19
Tularemia PPG
• Protection of NHP by vaccination with LVS lot 16
• Vaccination
– Unvaccinated control
– Subcutaneous 5 x 107 cfu
– Bronchoscopy 1 x 106 cfu
• SCHU S4 Challenge by bronchoscopy 42 d after
vaccination: target 1,000 cfu, inoculum ~140/lobe
• Survival :
– Unvaccinated: 0/2 survived (d 5 and 7)
– Subcutaneous: 2/3 survived (d 16)
– Bronchoscopy: 2/3 survived (8)
20
Action Items
•
•
•
•
•
Terry may order more nude rats and repeat the studies to understand the role of
T cells in passive immunization.
Terry is working on the 3rd draft of the rat passive immunity manuscript and hope
to submit to a journal by end of 2009.
Terry replot the human response data (correlate of protection) on a log scale
Rick, Patrick and Freyja: contemplate the lot of LVS to use for future NHP
vaccinations on the TVDC
Rick, Patrick and Freyja: contemplate using the Cerus /Aduro metabolic assay for
comparing lots of LVS or different treatments to the LVS, as another measure
beyond CFU recovered.
21