LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
8 April 2008
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
Milestones
#2
Active
Vaccinations of study personnel
#3
Active -
Optimization of bioaerosol methods
#4
Active -
Confirmation of aerosol in vivo in NHP
efficacy studies in primates
#7
Active -
SCHU S4 LD50 in primates
#12/13
Active -
Assays for detecting relevant immune
responses in animals and humans
#21
Active-
Correlates of protection- in vitro assay
or other readout of effector function of
Ft developed for multiple species
Slide 2
Milestone #2 – Vaccinations of Study Personnel

Vaccinations completed
–
32 LBERI scientists and staff received the LVS vaccination between
9/11/07 and 1/8/08

–
All have completed the 1 month post-vaccination follow-up
4 UNM scientists and staff received the LVS vaccination on 3/18/08

The 1 week post-vaccination follow-up was completed

The 1 month post-vaccination follow-up is pending
–
1 LBERI staff received the LVS vaccination on 4/1/2008
–
Dr. Lyons received UNM IRB approval to allow blood draws on the
vaccinated LBERI and UNM scientists after their LVS vaccinations. The
LBERI and UNM scientists and staff will be offered the opportunity to
volunteer to donate bloods for the development of immunoassays,
approximately 2 months after receiving the LVS vaccination
Slide 3
Milestone #2 – Vaccinations of Personnel
Plans for this month




4 UNM personnel awaiting 1 month follow-up
Continue follow-up with blood draws from vaccinated LBERI and UNM
personnel for the development of immunoassays
1 UNM personnel may be vaccinated on 4/29/08
USAMRIID is temporarily closing the LVS vaccinations until a new
protocol is in place (after 4/29)
Slide 4
MS#3 – Flow Diagram
MS 3: Bioaerosol Development
Collison Nebulizer
Aeromist
Micropump
Order & receive
instrument
Order & receive
instrument
Order & receive
instrument
Set up instrument
Set up instrument
Set up instrument
Frozen
LVS
Fresh
LVS
Lyophilized
LVS
Frozen
LVS
Fresh
LVS
Frozen
LVS
Fresh
LVS
Down Select for
SCHU S4 Generator
Frozen
Schu4
Red: completed
Green: in progress
Blue: steps in the milestone
Fresh
Schu4
Prepare bioaerosol SOP and
write MS completion report
Slide 5
Milestone #3 – Bioaerosol Development
Accomplishments




Completed Collison, Sparging Generator, Micropump and
Aeromist LVS testing (fresh vs. frozen)
Tested additional technologies (e.g., ultrasonic, Aeroeclipse II,
others) with BG spores; F. tularensis testing not pursued due to
difficult setup, practicality and/or no significant differences vs.
Collison
Completed frozen and fresh SCHU S4 testing with the Aeromist
and Collison
Performed pathogenicity study of LVS and SCHU S4 bioaerosols
in mice.
Slide 6
SCHU S4 Bioaerosol Data to Date


No bioaerosol optimization testing was performed in March 2008
LBERI is experiencing SCHU S4 growth issues in Chamberlains
–
Have pinpointed problem to Chamberlains broth as
cultures grow as expected on BCGA and QC results are
normal
–
Working in conjunction with UNM personnel to remedy
problem
 Requesting UNM-produced Chamberlains lot
–
Is it worthwhile to explore media optimization for SCHU
S4? This was performed for LVS, but not for SCHU S4.
Slide 7
Milestone #3 – Bioaerosol Development
Plans for this month


Continue MS Completion report
Determine problem(s) with SCHU S4 growth in Chamberlains as
soon as possible
Slide 8
MS#4 – Flow Diagram
MS 4: NHP Aerosol Confirmation
Aerosol Challenge
Approach
Naïve NHP
Challenges
Vaccinated NHP
Challenges
MS 3
Cohort 1
(n=2)
Vaccinated
NHPs available;
awaiting
completion of
naïve and LD50
challenges
Mouse
Challenges
Cohort 1
Cohort 2
Cohort 2
(n=2); to
follow
mouse
challenges
Cohort 3
NOTE: Chamberlains growth problems have
halted Cohort 3 mouse challenges two times
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 9
MS#4 Overview

Mouse studies
–

Objectives:

To verify that aerosolized SCHU S4 are virulent in mice
(concern that aerosol method may alter virulence)

To determine SCHU S4 deposition as to related to the
bioaerosol parameters; this will influence NHP exposure
procedures
Naïve NHP studies
–
Objectives:


To verify that aerosolized SCHU S4 are virulent in NHP (similar
concern as described for mice)
Vaccinated NHP studies
–
Purpose is to demonstrate protection in NHP previously vaccinated
with LVS
–
This study will follow determination of LD50 aerosol dose
Slide 10
Milestone #4 – Confirmation of Aerosol in vivo in NHP

Pathology report completed (see following 2 slides) for 2 naïve
primates infected with SCHU S4 in December 2007.
Slide 11
Pulmonary histopathology
from cynomolgus
macaques exposed to
aerosols of F. tularensis
SCHU S4. A, C, and E are
from A04339, and B, D, and F
are from A04344.
Arrowheads (A, B) indicate
primary foci of
pyogranulomatous to
necrotizing
bronchopneumonia. Small
arrows (C) indicate smaller
foci of pyogranulmatous to
necrotizing embolic
pneumonia detected only in
A04339. Large arrow (E)
indicates a colony of small
coccobacilli.
Slide 12
Histopathology of the
tracheobronchial lymph node,
spleen and lung from a
cynomolgus macaque (A04339)
exposed to aerosols of F.
tularensis. Arrowheads in the
lymph node (A) and spleen (B)
indicate foci of pyogranulomatous
to necrotizing inflammation. Small
arrows indicate foci of nasal
turbinate ulceration with
fibrinosuppurative rhinitis in the
nasal cavity (C).
Slide 13
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for next month

Further investigate SCHU S4 deposition
–
BALB/c mice
–
Aeromist vs. Collison
–
48h Chamberlains broth

–
High and low doses

–


Growth problems encountered
1 x 107 and 108 CFU/mL in generator suspension; should result
in countable colonies upon culture
n=5 per group followed by immediate Nx and lung culture on selective
media
Complete Micro report
Schedule follow-on NHP SCHU S4 bioaerosol challenge (n=2); late April/early
May
Slide 14
MS#7 – Flow Diagram
MS 7: NHP SCHU S4 LD50/ED50
Round 1 (n=4 NHP each dose)
5000 CFU Delivered
500 CFU
50,000 CFU
(Based on virulence study)
Round 2 (n=4 NHP each dose)
x CFU (TBD)
y CFU (TBD)
Round 3 (n=4 NHP each dose)
x CFU (TBD)
Red: completed
Green: in progress
Blue: steps in the milestone
y CFU (TBD)
LD50/ED50
Determination
Slide 15
MS#7 Overview

Objectives:
–
To determine the SCHU S4 LD50 aerosol dose in NHP
–
To observe and document the natural history of disease
through clinical observations and in-life/post-life sample (blood
and tissue) analyses
Slide 16
MS#7 Tentative Endpoints

The endpoints for each set of exposures will be clinical observations,
temperature monitoring, body weight records, gross necropsy, and
viable bacterial blood/tissue cultures
Slide 17
Milestone #7 – Confirmation of Aerosol in vivo in NHP
Plans for next month



Confirm LD50 dosing/challenge scheme
Confirm endpoints and schedule appropriate personnel
Confirm SCHU S4 bioaerosol method
–

Aeromist vs. Collison
Initiate ABSL-3 move-in and challenge dates
–
Attempt late May/early June for 1st cohort
Slide 18
Milestone #12/13 – Immune Responses in Animals
and Humans
Immunoassay Development and Comparisons in Animal Models
Choose PBMC
Purification Method
Choose PBMC
Freezing Method
Method chosen:
Purdue ListServ
Cerus
Phenotype Blood
and PBMCs
Test whether method
results in loss of B
cells
Red: completed
Green: in progress
Blue: steps in the milestone
Develop
Immunoassay
methodologies
IFNg
Proliferation
assay:
Works for
Con A and
LVS
ELISPOT
Plasma
IgG
ELISA
Plasma
IgA
ELISA
IFNg
Intracellular
Staining
Slide 19
Update on responsiveness of non-LVS-vaccinated
NHPs to LVS antigens ex vivo
Issue:



Non-vaccinated NHPs often respond to FF LVS by secreting IFNg
as detected by the ELISPOT assay?
We do not know how consistent these responses are because
we rarely bleed the non-LVS vaccinated NHPs more than once.
It is important to determine whether the responses we observe
are consistent so that we can be confident of their responses
when we screen them before vaccination or use in other
protocols.
Slide 20
An example of consistent responses: A04260
A04260
Cell Mean for IFNg Spots
200
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
160
120
80
40
NT
0
TUL26
TUL31
TUL 26 PBMCs plated at 1.33 x 106/ml; TUL31 PBMCs plated at 1 x 106/ml
Tul 26 1/14/08 ; Tul 31 3/27/08
Slide 21
An example of inconsistent responses: A04168
Cell Mean for IFNg Spots
350
A04168
300
250
200
150
100
50
NT
NT
0
TUL21
TUL23
TUL31
All plated at 1.33 x 106/ml
Tul 21 11/26/07; Tul 23 12/4/07; Tul 31 3/27/08
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
Slide 22
Proliferation is consistent even if IFNγ secretion is not
1500000
1200000
900000
A04168
Media
Con A
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
300000
0
TUL21
NT
600000
NT
Cell Mean for RLU small
1800000
TUL23
Tul 21 11/26/07; Tul 23 12/4/07
Slide 23
A semi-consistent response: A05477
Cell Mean for IFNg Spots
A05477
450
Media
LVS hk Hi
400
LVS hk Mid
350
LVS ff Hi
300
LVS ff Mid
250
LVS ff Lo
200
LVS hk Super
150
SCHUS4 hk Super
100
SCHUS4 hk Hi
50
NT
SCHUS4 hk Mid
NT
SCHUS4 ff Super
0
TUL21
TUL23
TUL31
SCHUS4 ff Hi
All cells plated at 1.33 x 106/ml; Is FF and HK LVS mixed up in TUL 21?
Tul 21 11/26/07; Tul 23 12/4/07; Tul 31 3/27/08
Slide 24
Proliferation is consistent: A05477
Media
Con A
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
1500000
1200000
900000
300000
NT
600000
NT
Cell Mean for RLU small
1800000
0
TUL21
TUL23
Tul 21 11/26/07; Tul 23 12/4/07
Slide 25
MS 12/13: Plans for the next month


Continue to test PBMCs frozen and thawed using the Cerus
protocol
Continue to work on optimizing the IFNγ ELISPOT assay
such that non-LVS vaccinated NHPs have low background
responses and LVS-vaccinated NHPs have antigen-specific
responses.
–
Test higher doses of HK LVS and lower doses of FF LVS
–
Test a range of doses of HK- and FF-SCHU S4
–
Prescreen newly purchased NHPS for dose responsiveness to
LVS and Schu 4 antigen preparations.
–
Test the same range of antigen doses on LVS-vaccinated NHPs
to determine minimal antigen preparations dose that provides
sensitive and specific response in IFNγ ELISPOT assay and
proliferation assay.
Slide 26
MS 21 (LBERI/UNM)
Correlates of protection: in vitro assay or other readout of
effector function of Ft developed for multiple species

Work out the conditions to detect intracellular IFNγ production
by flow cytometry in LVS-vaccinated NHPs
–

Order needed reagents (anti-CD28)
Moving the flow assay to Correlates of Protection MS 21
because this is not meant as a routine assay but will lead to a
more mechanistic understanding
Slide 27
Action Items











Trevor: will try growing SCHU S4 on BCGA plates first and then innoculate into liquid
Chamberlains (completed 4/21/08)
Kristin will provide the USARMIID growth conditions to UNM
Trevor will write the MS3 MSCR and associated SOPs
Bob share technicium particle aerosol study at next LBERI tech call or at the site visit on
4/24/08
Julie W- tell Barbara the number of NHP to be vaccinated on the natural history study
Vicki- try to obtain clinical LVS lot prior to next NHP vaccination at LBERI
Kristin: has USAMRIID histopathology photos that she could share with Julie Hutt, from NHP
with tularemia showing riddling of white foci in spleen
Kristin can share the clinical signs detected at USAMRIID NHP infected with SCHU S4
Bob: will schedule NHP rooms longer and get IACUC amended for 35 days so NHP studies can
go as long as the USAMRIID NHP studies.
Vicki/Freyja: NIAID should supply both SCHU S4 and LVS O antigen mutants to LBERI to test
impact on background in Elispot assay with non vaccinated NHP. Will send to UNM.
Julie: Under MS21, will try to develop the triple positive functional T cell intra cellular flow assay
in NHP
Slide 28