Progress Report
12/02/2009
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:
Effect of multiple serum treatments on protection
12/13. Immunoassays:
Staining LVS for microagglutination assay
21. Correlate of protection assay using human PBMC
New data from prevaccination samples and UBS samples
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 serum dose or with
repeated serum treatment
• Protection eliminated by depletion of CD8 T cells and in nude
rats
• Completing experiments for manuscript
4
Repeated Serum Treatment Did Not
Reduce Bacterial Burden
Questions: Repeatable? Any effect before day 7?
5
Repeated Serum Treatment Did Not
Reduce Bacterial Burden Compared with
Single Treatment
Lung
Spleen
Liver
Total CFU (Log10)
10
10
10
8
8
6
6
4
4
4
2
2
2
0
0
0
8
6
0
3
6
9
12
Days Post-challenge
15
0
3
6
9
12
Days Post-challenge
15
NRS
IRS (d -1)
IRS (d -1, 1, 4, 7, 10, 13)
0
3
6
9
12
15
Days Post-challenge
6
Milestone 11 Humoral Immunity
(in progress)
• Complete experiment with multiple serum treatment
• Confirm CD4 inactivation by functional assay
• Repeat passive immunization/challenge in T cell
deficient nude rats
• Continue manuscript preparation
7
Splenomegaly in Passively Immunized Rats after
SCHU S4 Challenge
8
Milestone 11 Cellular Immunity: Plans
• Develop method for adoptive transfer of immune T
cells into naïve rats (technique reported for Fischer
344 rats) – rats vaccinated
9
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
10
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
11
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
• Current goal: Increase sample size (unvaccinated and
vaccinated ) to determine statistical significance
12
Accumulated Results from
Human PBMC Studies – end of 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
13
Accumulated Results from
Human PBMC Studies – end of Nov 2009
CFU -fold increase with
48-hour FF-LVS pretreatment,
normalized to no-pretreatment control
1.25
-fold increase, normalized to control
2-tailed Mann-Whitney nonparametric
p=0.0759
18.1 (UBS)
1.00
35 (prevax 8.1)
0.75
31 (prevax 6.1)
0.50
27.2 (UBS)
0.25
33
0.00
32 (UBS)
18.2
36 (prevac 9.1)
27.1 (UBS)
29 (prevac 4.1)
34 (UBS)
20
30 (prevax 5.1)
26.1
vaccinated (N = 4) unvaccinated (N = 9)
14
Optimization to Reduce Variability Among
Unvaccinated samples: Ag titration
FT-AH-35
prevax 8.1 PBMC
FF-LVS #6
FT-AH-34
UBS PBMC
1. FF-LVS #6 titration
1.010 8
1.010 8
control (no pretreatment) (3870x increase)
FF-LVS#6 5.0% (340x increase)
FF-LVS#6 0.5% (940x increase)
FF-LVS#6 0.05% (4510x increase)
1.010 7
1.010 7
1.010 6
1.010 6
total CFU / well
total CFU / well
control (no pretreatment) (1350x increase)
FF-LVS#6 5.0% (1210x increase)
FF-LVS#6 0.5% (1960x increase)
FF-LVS#6 0.05% (1770x increase)
1.010 5
1.010 5
1.010 4
1.010 4
1.010 3
1.010 3
1.010 2
1.010 2
0
24
48
72
HOURS POST-INFECTION
0
24
48
72
HOURS POST-INFECTION
15
Planned Experiments
• Compare FF-LVS from Lyons lab and newly arriving
DVC stocks
• 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
16
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
• Test Lm-based vaccines from Aduro in mice and rats
for protection against SCHU S4 challenge
– BH2172 (Live Lm677 KatG-SL8)
– BH2182 (Live Lm677 IglC-B8R)
18
Vaccination with rLM/iglC Protected Mice
Against i.n. LVS Challenge (UCLA)
i.d. immunization – 0 and 4 weeks 106 Lm strains/mouse, 104 LVS/mouse
i.n. challenge – 8 weeks
4400 cfu
4.5 x 104 cfu
19
Vaccination with rLM/iglC Protected Mice
Against Aerosal SCHU S4 Challenge (UCLA)
i.d. immunization – 0 and 4 weeks 107 Lm strains/mouse, 104 LVS/mouse
Aerosol challenge – 10 weeks
20
Experimental Design
21
Intravenous Vaccination with Lm-based Vaccines
Protected Mice Against i.v. LVS challenge but not i.n.
SCHU S4 challenge (Aduro/UNM)
Immunization (0 wk) – 3 x 105 Lm strains/mouse i.v. & 4 x 103 LVS/mouse i.n.
Boost (6 wk) -- 2 x 106 Lm strains/mouse i.v. & 5 x 103 LVS/mouse i.n.
Challenge (10 wk) – 1.52 x 105 LVS i.v. and ~80 SCHU i.n.
n = 10 / group
i.v. LVS challenge
i.n. Schu4 Challenge
100
Percent survival
Percent survival
100
80
60
40
20
0
80
PBS
LVS
BH2172 (Lm677 KatG-SL8)
BH2182 (Lm677 IglC-B8R)
60
40
20
0
0
5
10
Days post challenge
15
0
5
10
15
Days post challenge
22
Milestone 10: Plans
• Repeat mouse vaccination/challenge with
lower SCHU S4 dose and i.v. route?
• Challenge vaccinated F344 rats
23
Others
• MS 12/13: Immunoassays
– Microagglutination assay
• Stained LVS lot 9 with hematoxylin
• Estimate enough for 4 x 96 well plates
• Titer Ag with positive control rabbit serum – may need more lot 9
LVS
• Develop assays with sera from human, rat, mouse
• MS 5: model development
– F344 rats infected with 5 x 104 SCHU S4 developed
bacteremia within 2 d of infection
– Tested persistence of immunity
24
LVS-induced Immunity Maintained 3 mo
after LVS Vaccination
Challenge dose = 3.5 x 104 cfu/rat
Percent survival
100
80
60
Naive (n = 6)
LVS vaccinated (90d; n = 5)
40
20
0
0
5
10
15
20
25
Days post challenge
25
• Terry will grow Lot 9 LVS from the one
remaining lot 9 LVS vial and make more
stained antigen preparation for the
microagglutination assay.
• Barbara will ask USAMRIID how grow lot 9
LVS. Was Mueller Hinton Broth used?
26