Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 1 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Signature Page
Author’s Signature:
_Justin Skoble_____________________
Typed Name of Author
Acceptance by Subcontracting Institution:
_Suzanne Margerum_______________
Typed Name of Subcontracting PI
Acceptance by the University of New Mexico:
_Rick Lyons______________________
C. Rick Lyons, MD. PhD
Acceptance by NIAID:
_Freyja Lynn and Patrick Sanz________
Typed Name of NIAID Project Officer
_Heidi Holley______________________
Typed Name of NIAID Contract Officer
___2/2/2010__
Date Accepted
Page 1 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 2 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Table of Contents
1
2
3
4
Milestone Summary ...................................................................................................................................................2
Milestone Objectives..................................................................................................................................................3
Methods, Critical Reagents and SOPs........................................................................................................................3
Salient Original Data, Results, Interpretation, Quality Control .................................................................................5
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location annotations) .................5
4.2
Interpretation ............................................................................................................................................ 10
4.3
Quality Control ......................................................................................................................................... 11
5 Deliverables Completed ........................................................................................................................................... 11
6 Appendices ............................................................................................................................................................... 12
6.1
Appendix 1: Original Data Tables and Figures ....................................................................................... 12
6.2
Appendix 2: Quality Assessment of Milestone Completion and Report ................................................. 14
6.3
Appendix 3: Additional Data/Figures not included in the Text of the Milestone Completion Report
(Section 4) .............................................................................................................................................. 16
1
Milestone Summary
The original goal of MS42 was to determine whether killed but metabolically active (KBMA)
Francisella tularensis (Ft) subspecies. novicida vaccines induced a cellular immune response that
protected against wild-type Ft novicida challenge in mice. Cerus planned to measure the level and
duration of protection against a Ft novicida challenge and thus evaluate whether KBMA Ft novicida
could function as a potent vaccine.
KBMA Ft novicida uvrB vaccine stocks produced in MS41 were tested in mice to determine
whether they provided protection against a 100 x IP LD50 challenge of wild-type Ft novicida
(U112). KBMA Ft novicida uvrB were 100% protective when a single dose was administered at
or near the LD50 of the KBMA vaccine (1 x 109 IP, 1 x 108 IV); at doses below the LD50, a single
vaccination was less effective. 100% protection was also achieved by administration of an
approximately 0.1 IV LD50 dose (1 x 107 KBMA particles) when the vaccine was given twice
separated by 3 weeks. In these studies, heat-killed Ft novicida protected as well as the KBMA Ft
novicida vaccine, and was found to be less toxic to the mice.
Since the KBMA Ft novicida uvrB vaccine did not perform better than the heat-killed Ft novicida
vaccine, cellular and humoral mechanistic questions were pursued. Mice were vaccinated with
KBMA Ft novicida uvrB vaccine and then depleted of different subsets of T cells prior to lethal
challenge with wild type Ft novicida. 100% of the mice vaccinated with KBMA Ft novicida
uvrB and 100% of the mock depleted mice survived the lethal challenge, but depletion of CD4+
T cells decreased the survival rate to 80%, depletion of C8+ T cells had no effect, and surprisingly,
90% of mice depleted of both T cell populations survived a lethal challenge with wild type Ft
novicida. Together, these data demonstrated that CD4+ T cells marginally contribute to a
protective immune response in a non-CD8+ T cell-dependent manner. One possible mechanism by
Page 2 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 3 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
which CD4+ T cells can contribute to immune responses is by providing help to B cells. In support
of this interpretation, when CD4+ cells were depleted from mice vaccinated with KBMA Ft
novicida their anti-Ft titers were reduced compared to mock depleted control animals.
Furthermore, ina passive transfer experiment high-titer anti-Ft sera from KBMA vaccinated
animals provided some protection against a lethal wild-type Ft novicida challenge. Together, these
data suggested that the KBMA Ft novicida uvrB vaccine may be inducing a humoral immune
response. Because heat-killed Ft novicida protected as well as KBMA uvrB, we assumed this
was due to the injection of surface antigens (e.g. LPS) and induction of a humoral immune
response and not as a function of metabolic activity or the ability to deliver secreted antigens.
Thus, we saw limited value in continuing with development of KBMA Ft novicida vaccine
candidates in this milestone or in Milestone 43.
This milestone was terminated when only approximately 25% complete in order to pursue a
strategy of Listeria monocytogenes based vaccines that induce cell-mediated immune responses
against Ft antigens.
2
Milestone Objectives
From the original Statement of Work: Milestone 42 will assess the ability of the
photochemically treated Ft novicida uvrA or uvrB to protect against a lethal dose of virulent Ft.
novicida. This MS 42 MSCR reports on the protective efficacy of KBMA Ft. novicida uvrA or
uvrB as a proof of principle for this platform technology.
3
Methods, Critical Reagents and SOPs
Mouse studies. 6-12 week-old-female BALB/c mice were obtained from Charles River
Laboratories (Wilmington, MA). The mice were housed in individually HEPA-filtered cages with
autoclaved bedding. Food and water were supplied to the mice ad libitum. Animals were allowed
to acclimate to their surroundings for at least 1 week prior to use. Animals were injected SC, IP, or
IV via the tail vein, with 100 µl diluted thawed KBMA suspension as described previously
(Brockstedt, D.G. et al., Proc Natl Acad Sci USA. 2004; 101(38):13832-7). LD50s were calculated
using the method of Reed and Muench (Reed, L. J., and H. Muench. 1938. Amer Journ Hygiene
27:493) and the LD50 of U112 in mice was reported in the MS40 MSCR ver 1.0 of 3/3/2009 (see
Table 1 on page 9 of 20). One month after vaccination, animals were challenged via the IP route
with 100 x the LD50 (approximately 100 cfu from diluted thawed U112 cell bank, which was
verified by plating 100µl of injectant directly on CHAH plates in triplicate) with the wild-type Ft
novicida. Animals were monitored for survival. Four weeks after lethal challenge, blood was
collected from surviving animals by terminal heart puncture and serum was separated by
centrifugation in serum separator tubes. Serum was transferred to naïve mice via IV injection, and
these mice were challenged as above. All animals were treated according to National Institutes of
Page 3 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 4 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Health guidelines, and protocols requiring animal experimentation received prior approval from the
Cerus Animal Care and Use Committee.
Handling of bacteria. Cultivation of Ft novicida strains was performed at 37oC using Cystine
Heart Agar (Difco) supplemented with 10% bovine hemoglobin (Becton, Dickinson) as solid
medium (CHAH) for colony enumeration following PRP01.002. For liquid cultivation of Ft
novicida strains, individual colonies were inoculated into Chamberlain’s defined medium (CDM)
and incubated at 37oC with shaking at ~200 RPM. For preparation of CDM liquid media, 24.2
grams premixed formulation (Teknova cat# C0715) is added to water, the pH is adjusted to 6.26
and sterile filtered through 0.22 m filters.
Whole-bacteria ELISA. The antibody response against Ft was measured by enzyme-linked
immunosorbent assay (ELISA) using minor modification to standard procedures (Koskela, P and E.
Herva. 1982. Infect. Immun.36 (3): 983. The 96-well maxiSORP plates (Nunc) were coated with
serial dilutions of KBMA Ft novicida in 0.1 M carbonate buffer (pH 9.6) overnight at 4°C and then
washed with PBS containing 0.05% (vol/vol) Tween 20 (PBST). Plates were then blocked with 5%
(wt/vol) skim milk in PBS for 1 h. Serum samples were pooled by group, and pools were diluted in
PBST and added to triplicate wells and incubated at room temperature for 1 h and washed with
PBST. Bound antibody was then detected using horseradish peroxidase (HRP)-conjugated
polyclonal goat anti-mouse IgG (Jackson Immunoresearch) diluted 1:10,000 in wash buffer and
incubated for 2 h. Plates were developed with 100 µl of TMB substrate (Calbiochem) for 5-10
minutes. The reaction was stopped by the addition of 50 µl 1M sulfuric acid, and the OD450 of the
plates were measured using a SPECTRAmax plus 384 plate reader (Molecular Devices). A
concentration of 5.12 x106 KBMA particles was empirically found to provide the optimal signal to
noise ratio. Antibody titers were expressed as the maximum dilution of sample giving an
absorbance of more than 3 x the absorbance due to nonspecific binding detected in blank wells
without serum.
List of Critical Reagents
Cystine Heart Agar (Difco)
Bovine Hemoglobin (Becton, Dickinson)
Chamberlains Defined Medium (Teknova cat# C0715)
BALB/c mice (Charles River Laboratories. Wilmington, MA)
∆uvrB Ft novicida (UTSA KKF71 strain; MS#39)
U112 wild type Ft novicida (UTSA)
SOP Number1
FAP-01.001
FAP-01.003
SOP Title
Animal Facility Gowning
Animal Handling and Restraint
Page 4 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 5 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
SOP Number1
FAP-01.026
FAP-01.028
QCP-01-003
Accepted Date: 5/5/09
SOP Title
Intravenous (IV) Injections
Intraperitoneal (IP) Injections
Determination of Viable Bacterial Count of a Bacterial Culture
1
Individual Standard Operating Procedures will be reviewed separately and accepted by the Subcontracting PI and UNM
PI NOTE: The above 5 procedures were accepted by UNM and NIAID in association with Cerus MS 40 MSCR and are
not provided in conjunction with the MS 42 MSCR.
4
Salient Original Data, Results, Interpretation, Quality Control
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location
annotations)
Rationale. Our ultimate goal was to produce a Killed But Metabolically Active (KBMA)
Francisella tularensis (Ft) vaccine. KBMA vaccines are produced by a process of
photochemical inactivation of organisms using a synthetic psoralen (S-59) and ultraviolet
light (UVA). The ultraviolet light activates the S-59 to crosslink nucleic acids and prevents
the replication of the bacterial chromosome. Photochemical inactivation is a process used
commercially by Cerus for pathogen inactivation in plasma and platelet products for
transfusion. Nucleotide excision repair (NER) is the primary mechanism of repair of
crosslinked DNA. This NER process is initiated by the exinuclease complex composed of the
products of the uvrA, uvrB, and uvrC genes. While a broad range of wild-type pathogens can
be killed by photochemical inactivation, bacteria that are engineered to be NER deficient are
typically extremely sensitive to the process, and replication can theoretically be prevented
with a single crosslink. Photochemical inactivation with randomly distributed infrequent
crosslinks allows the NER-deficient bacteria to maintain a high degree of metabolic activity
while preventing replication leading to a state we refer to as KBMA. In MS41 we produced
KBMA Ft subspecies novicida uvrB vaccine stocks. The goal of this milestone was to
determine whether KBMA Ft novicida uvrB vaccine stocks produced in MS41 were capable
of providing protection against a lethal wild-type Ft novicida challenge. Ft novicida were
proposed in this milestone in order to rapidly demonstrate proof of the KBMA concept in the
mouse model of tularemia because Ft novicida is easier to genetically manipulate than LVS
or type A strains of Ft.
A single high dose of KBMA Ft novicida vaccine can confer protection against a lethal
challenge of wild-type Ft novicida. In Milestone 41 it was demonstrated that KBMA Ft
novicida uvrB was dramatically attenuated for virulence when administered by the SC, IP,
or IV routes (Cerus Animal study AS07-009). One month after a single vaccination on MS
41, animals surviving inoculation with 1x108 or 1x109 particles were then challenged on
MS42 with 100 x IP LD50 of wild type Ft novicida to determine whether a single
immunization with the KBMA vaccine induced a protective immune response (T-1).
Page 5 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 6 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Animals that were vaccinated with vehicle alone all died within 3 days of challenge. Animals
that survived a single IP vaccination with 1x 109 KBMA or heat-killed Ft novicida uvrB,
were fully protected, however only 60% of the animals that received a 1x108 dose of KBMA
IP survived. None of the animals vaccinated IV with 1x109 KBMA Ft novicida uvrB
survived the immunization and only 2 of 5 survived IV immunization with 1x108, but both of
these mice then survived the subsequent lethal challenge with wild-type Ft novicida. While
vaccination with the KBMA Ft novicida uvrB via the SC route was less toxic (all of the
mice in the groups that received SC immunization survived), none receiving the 1x108 dose
and only 60% receiving 1x109 dose survived the subsequent lethal wt challenge. Together
these data suggest that a very high single dose of KBMA Ft novicida is required to induce
protective immunity, and that this level appears to be near the LD50 for each route
(approximately 1 x 109 IP, 1 x 108 IV, >109 for SC). It is also important to note that the heatkilled control administered at 1 x 109 IP was avirulent at this dose, and also was 100%
protective against the lethal challenge. These data suggest that, by these routes of
administration, an unacceptably high dose of KBMA vaccine needs to be administered to
confer protection after a single vaccination without further dose regimen optimization (e.g.
boost vaccination).
T-1
AS07-009
HBSS
HK Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
Vaccinatio
n dose
(particles)
1.00E+09
1.00E+09
1.00E+08
1.00E+09
1.00E+08
1.00E+09
1.00E+08
Route
IV
IP
IP
IP
IV
IV
SC
SC
Vaccination
Survivors
5 of 5
5 of 5
2 of 5
5 of 5
0 of 5
2 of 5
5 of 5
5 of 5
100 x IP LD50
Challenge
Survivors
0 of 5
5 of 5
2 of 2
3 of 5
NA
2 of 2
3 of 5
0 of 5
Protection
Mean Time
to Death
0%
100%
100%
60%
NA
100%
60%
0%
3d
NA
NA
4d
NA
NA
4.5d
4d
T-1 Survival of Balb/c mice after vaccination with a single dose of KBMA Ft novicida
uvrB and lethal IP infection with wild-type Ft novicida. Balb/c mice were injected with
KBMA Ft novicida uvrB or buffer, and survivors were challenged one month later with
100 cfu of wild-type U112 by IP injection and monitored for survival. HK annotates “heat
killed”.
A prime-boost regimen with 0.1 IV LD50 KBMA induces protective immunity against a
lethal wild-type Ft novicida challenge in a CD8+ T cell independent manner. After
showing that an excessively high single dose of KBMA Ft novicida was required for
protection, we next wanted to determine whether two lower doses of KBMA vaccines could
protect against lethal Ft novicida challenge. Entering the TVDC contract, Cerus had
assumed that KBMA Ft novicida would induce protective T cell response similar to KBMA
Page 6 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 7 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Listeria monocytogenes, but thereafter Cerus determined that heat killed Ft vaccines also
conferred protection, and heat killed vaccines usually only induce humoral immunity. Thus
Cerus wanted to determine whether the protection afforded by KBMA Ft novicida vaccines
was T cell mediated by performing cellular depletion studies. We vaccinated Balb/c mice
twice (separated by 3 weeks) with 1 x 107 KBMA particles (approximately a 0.1 LD50 dose),
or buffer alone. Three days prior to a lethal challenge, vaccinated animals were injected daily
with 250 µg antibodies that deplete CD4+ cells, CD8+ cells or the same amount of both
antibodies to deplete both types of cells. This concentration of antibody, and regimen has
been used previously by Cerus to deplete T cells and abrogate protection induced by L.
monocytogenes vaccination and was confirmed by flow cytometry (Bahjat et. al, Infect.
Immun., 2006, p. 6387-6397, Vol. 74, No. 11, and Bahjat et al J. Immunol., 2007, 179: 73767384). All animals that were vaccinated with vehicle (HBSS) alone died within 3 days of
100 x IP LD50 challenge. All animals that were vaccinated twice with KBMA Ft novicida
and not depleted of T cells survived the lethal challenge (T-2). Surprisingly, 100% of the
animals that were vaccinated with KBMA Ft novicida and were then depleted of CD8+ T
cells also survived, suggesting that CD8+ T cells do not contribute to protection in this
model. After 6 to 7 days following 100x IP LD50 challenge 2 mice in the CD4 depleted group
died and 1 mouse in the CD4 and CD8 depleted group died, suggesting a strong degree of
protection was maintained despite the depletion. These data suggest that CD4+ T cells play a
minor role in protection during the lethal challenge, and since depletion of CD8+ T cells in
the double depletion group did not have a compound adverse effect on survival, that the
CD4+ T cells are contributing to protection in a CD8 independent manner. Together these
data suggest that the mechanism of protection after KBMA Ft novicida vaccination was
likely to be antibody mediated and that it was possible that the CD4+ T cells were providing
help to B cells and increasing the potency of the humoral immune response. To test this
further, sera were collected from the surviving animals by terminal cardiac puncture and
tested for anti-Ft titer and used in passive transfer experiments.
T-2
Vaccination
HBSS
KBMA uvrB
No Depletion
0% (MTD 3d)
100%
CD4
80% (MTD 6.5d)
CD8
100%
CD4 + CD8
90% (MTD 7d)
T-2 Survival of mice vaccinated with two doses of KBMA Ft novicida uvrB and
challenged with 100 x IP LD50 of U112 after depletion of T cell subsets. 40 Balb/c mice
were vaccinated IV with 1 x 107 KBMA Ft novicida uvrB and 10 mice were vaccinated with
buffer. 3 weeks after the first vaccination, all animals were boosted with the same. 4 weeks
after boost vaccination 10 animals per group were injected IP with antibodies to deplete CD4+
cells, CD8+ cells or CD4+ and CD8+ cells. Animals were depleted of T cell populations for 3
consecutive days prior to a 100 x IP LD50 Ft novicida challenge and then monitored for
survival. Animal Study number AS07-017.
Page 7 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 8 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Vaccination with KBMA Ft novicida induces a humoral response against Ft novicida.
Since T cell depletion had only a modest effect on protection after lethal challenge with wildtype Ft novicida (in study AS07-017), Cerus wanted to determine whether survival in this
study correlated with magnitude of humoral immune responses. Thus, Cerus drew blood
from the surviving animals 3 weeks after the challenge and used naïve animals as controls
since the buffer vaccinated animals all succumbed to the lethal challenge. A whole-bacteria
ELISA was used to measure the titer of antibodies against Ft novicida that were found in
serum of mice. KBMA Ft novicida were used to coat wells of microtiter plates at a
concentration of 5.12 x106 particles per well. Sera from naïve mice or surviving mice from
AS07-017 (that had been vaccinated twice with KBMA Ft novicida uvrB, depleted of T cell
populations or mock depleted and subsequently challenged with a 100 x LD50 dose of U112)
were adsorbed to the killed bacteria and detected with a goat anti-mouse HRP-conjugated
antibody. As shown below (F-1), naïve mice had almost no anti-Ft titer and all vaccinated
animals had significant anti Ft antibodies. Of the vaccinated animals, the animals that were
depleted of CD8+ T cells had the highest anti-Ft titer. The animals with the lowest anti-Ft
titer came from animals depleted of CD4+ T cells which is the group that was the least well
protected against lethal challenge. This finding is consistent with the ability of CD4 cells to
provide help in the form of cytokine secretion to B cells that produce antibodies. However,
the antibody titer from the doubly depleted group was very similar to the mock depleted
group and the survival was 90% rather than 100% thus the CD4 T cells may contribute in a
very modest way to protection via a mechanism that does not result in increasing antibody
titer.. These sera were then used for a passive transfer study to determine whether the anti-Ft
antibodies present were able to confer protection against a lethal Ft novicida challenge.
Page 8 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 9 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
F-1
Anti-FT ELISA
10000
Titer
1000
100
10
1
Naive
Mock
depleted
CD4
depleted
CD8
CD4+CD8
depleted depleted
F-1 Whole-bacteria ELISA to determine the titer of anti-Ft antibodies in serum of
KBMA vaccinated mice. Microtiter plates were coated with 5.12 x106 particles of KBMA
Ft novicida and were then incubated with serial dilutions of pooled sera from naïve mice or
mice that had been vaccinated with KBMA Ft novicida, depleted of T-cell populations,
survived a lethal challenge in Animal Study number AS07-017. Bound anti-Ft antibodies
were detected using a goat anti-mouse antibody conjugated to HRP and TMB. Antibody
titers were expressed as the maximum dilution of sample giving an absorbance of more than 3
x the absorbance due to nonspecific binding detected in wells without serum. The pooled
sera from each group was tested in duplicate and the titers were calculated as the mean of the
absorbance at 450nM times the dilution factor (dilutions reported here were 1:100 for Naïve
animal serum, 1:25600 for mock depleted, CD4 depleted, and CD4 and CD8-depleted animal
sera, and 1:51200 for CD8 depleted animal serum. Absorbance values ranged from 0.128 to
0.194) . The error bars represent the standard deviation between replicates. Notebook: 980page031.
Passive transfer of sera from animals vaccinated with KBMA Ft novicida confers some
protection against a lethal Ft novicida challenge. To test directly whether the mechanism
of protection provided by KBMA Ft novicida vaccination is humoral, we collected serum
from each of the vaccinated animals that survived the lethal challenge in AS07-017, and also
from 10 naïve mice. The sera were pooled by group, and 200 µl of serum was injected IV
into 5 Balb/c mice per group 1 day before, and 100µl of serum 4 hours before a 100 x IP LD50
challenge dose of U112 was administered. The anti-Ft titers of each group are shown in F-1.
After the challenge, the animals that received serum from naïve mice all died within 3 days
(F-2). On the other hand, at 3 days post challenge, all the mice that had received sera from
Page 9 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 10 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
KBMA vaccinated mice were still alive. Ultimately only one mouse of 20 was protected by
the passive transfer of sera from KBMA vaccinated mice, but the delay in the time to death
compared with mice that received naïve mouse serum was significant (p value 0.0014). The
animal that survived was in the group that received serum from CD8-depeleted mice which
had the highest anti-Ft titer. If we had been able to transfer more serum, more mice may
have survived the lethal challenge (as reported previously by Bernard Arulanandam’s group
at UTSA). These data suggest that humoral immunity plays a role in protection of KBMA Ft
novicida vaccinated mice against a lethal wild-type Ft novicida challenge. We demonstrated
previously that protection was also observed following vaccination with heat-killed Ft
novicida.
F-2
AS07-045 Survival After Pasive Transfer of Serum
Percent survival
100
Naive serum
KBMA uvrB immunized
KBMA imunized -CD4
KBMA imunized -CD8
KBMA imunized -CD4/-CD8
80
60
40
20
0
0
5
10
15
Day
F-2. Survival of passively immunized mice after 100 x IP LD50 challenge with wild-type
Ft novicida. 5 Balb/c mice per group were injected IV with 200µl of pooled serum from
animals that were immunized with KBMA Ft novicida in AS07-017, or with serum from
naïve mice on day -1. Animals were passively immunized again with 100µl of serum IV 4
hours before a 100 X IP LD50 challenge dose of wild-type Ft novicida was administered IP.
Animals were monitored for survival for 2 weeks. Animal Study number AS07-045.
4.2
Interpretation
We demonstrated that KBMA vaccines were able to protect mice against a 100 x IP LD50
challenge of wild-type Ft novicida. To achieve protection after a single vaccination,
extremely high doses (at or near the LD50 of the vaccine) were required. At these doses, heatkilled Ft novicida also provided protection and were not virulent. We also achieved 100%
protection after administration of a 0.1 IV LD50 of KBMA Ft novicida by administering the
vaccine twice separated by three weeks. We then demonstrated that this protection was
independent of CD8+ T cells and was only slightly reduced in the absence of CD4+ T cells
suggesting that the KBMA vaccines induced a humoral immune response. This was
Page 10 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 11 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
confirmed by measuring the titer of antibodies that recognized whole bacteria. Passive
transfer of a small amount of serum from KBMA-vaccinated mice was able to significantly
extend the time to death of most mice and protect one. Together these data suggest that
KBMA Ft novicida vaccines may have induced a humoral response that protected mice from
a lethal challenge of wild-type Ft novicida but that the KBMA vaccine was not superior to
heat-killed Ft novicida.
Heat-killed vaccines often induce humoral immunity to bacterial surface antigens. Since
even heat killed Ft novicida protected in the Ft novicida challenge model (something we did
not expect to see when this project was initiated), there was no apparent added value of a
KBMA Ft novicida vaccine. Indeed this observation also led Cerus to re-evaluate the KBMA
Ft strategy entirely. We concluded there was little value in screening additional attenuated
KBMA double mutant vaccines in MS43 as well because we now predicted that all of the
attenuated mutants would protect in this assay (like heat killed). If each double mutant
protected fully against a lethal challenge, then it would be difficult to to rank attenuated
KBMA double mutants using the Ft novicida protection model(the goal of MS 43). Thus,
Cerus did not initiate Milestone 43. Because the KBMA Ft novicida vaccine did not perform
better than heat killed and did not induce a protective cellular immune response, Cerus
proposed to terminate this milestone when only 25% complete. UNM and NIAID supported
the termination of MS 43. Cerus focused new efforts instead on developing live-attenuated
and KBMA Listeria monocytogenes vaccine that expressed Ft antigens in order to induce a
cellular immune response in new Milestones 55-59.
4.3
5
Quality Control
 Each experiment was documented in a laboratory notebook that was read, understood and
countersigned by an individual who did not participate in the experiment.
 Analysis of survival data was carried out using the Kaplan-Meier method, and the log rank
test was used to determine the statistical significance of observed survival differences
using Prism software (GraphPad).
 Naïve animals or naïve sera were used as controls in each experiment.
Deliverables Completed
From statement of work: Report documenting ability of KBMA F. novicida uvrA or uvrB, to protect
against virulent F.novicida. MS 42
-The MS 42 milestone completion report serves as the report due as a deliverable.
Page 11 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 12 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Deliverable Reagents
Bacterial
Strain
# of
vials
Vial
Concentra
-tion
Storage
media
Date Stored
or Date
Transferred*
Storage location*
Date Stored
or Date
Transferred*
Storage location*
(Institution, room, shelf, etc)
NA
Tissue
identifier
# of
blocks
Tissue
Type
~ Mass
per
block
(Institution, room, shelf, etc)
NA
RNA/DNA
# of
vials
Vial
Concentra
-tion
Storage
media
Date Stored
or Date
Transferred*
Storage location*
#
Concentra
-tion
Storage
media
Date Stored
or Date
Transferred*
Storage location*
(Institution, room, shelf, etc)
NA
Polypeptide
(Institution, room, shelf, etc)
NA
*The storage location should allow a future researcher to specifically find the stored reagent.
When the “storage location” is equal to the creator’s location, enter the “Date Stored” in the “Date
Stored or Date Transferred” column. When the “storage location” indicates that the reagent has
been transferred to another institution, enter the “Date Transferred” in the “Date Stored or Date
Transferred” column.
6
Appendices
6.1
Appendix 1: Original Data Tables and Figures (those included in the above
sections 3 and 4)
Table/
Figure1
Title
T-1
Survival of Balb/c mice after vaccination with a
single dose of KBMA Ft novicida uvrB and
lethal IP infection with wild-type Ft novicida
Notebook
Location2
Electronic Location2
(Full Path & File Name)
(Notebook # and
page numbers)
AS07-009
AS07-009 KBMA FTn LD50
Summary C:\Documents and
Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\animal data
Page 12 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 13 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Notebook
Location2
Accepted Date: 5/5/09
Electronic Location2
Table/
Figure1
Title
T-2
Survival of mice vaccinated with two doses of
KBMA Ft novicida uvrB and challenged with
100 x IP LD50 of U112 after depletion of T cell
subsets.
AS07-017
AS07-017 Summary
C:\Documents and
Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\animal data
F-1
Whole-bacteria ELISA to determine the titer of
anti-Ft antibodies in serum of KBMA
vaccinated mice.
NB980 p.031
980-031: C:\Documents and
Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\francisella papers\Data
F-2
Survival of passively immunized mice after 100 AS07-045
x IP LD50 challenge with wild-type Ft novicida.
(Full Path & File Name)
(Notebook # and
page numbers)
AS07-045 survival data:
C:\Documents and
Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\francisella papers\Data
Use abbreviation “T” for table and “F” for Figure (e.g. T-1 for table 1 or F-3 for figure 3)
If the data location has changed relative to the location reported in the original monthly
technical report, then provide both the previously reported data location and the final data location
1
2
Page 13 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 14 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
6.2
Accepted Date: 5/5/09
Appendix 2: Quality Assessment of Milestone Completion and Report
Assessment Criteria for Milestone Completion
Evaluation of Milestone Completion Report
Yes No N/A Comment
Has the Milestone Completion Report format been used and all
sections completed, including Milestone Summary, Milestone
Objectives, Methods Reagents & SOPs, Salient Original Data
Results Interpretation & Quality Control, Deliverables
Completed, and Appendices?
Does the Milestone Summary include the milestone’s goals,
milestone results, an overall interpretation of the milestone’s
data and conclusion?
Do Methods, Critical Reagents and SOPs include summarized
methods and details necessary to re-perform critical
experiments? A list of critical reagents? The completed table of
SOPs?
X
Are salient negative and positive original data included in the
Milestone Completion Report?
Has the Deliverables Table been completed?
Have the Appendices been completed?
Are the specific original data associations with experiments
clearly annotated in the “Salient Technical Data” section of the
Milestone Completion report?
X
Evaluation of Data included
X
X
No new SOPs were
generated for this
milestone, Standard
published methods or
methods reviewed in
association with the MS
40 MSCR were used on
MS 42
X
X
X
Yes No N/A Comment
Are the salient original data and results included in an
organized, easily interpretable format?
Is the rationale included?
Do tables and figures have legends and original data location
annotations?
Is the data interpretation clear?
Is the data storage location listed in Appendix 1 sufficient for
data retrieval in the future? (E.g. notebook numbers and pages,
electronic file locations including directory paths and file
names). Are prior data locations cross-referenced to final data
locations?
X
Is the data backed up electronically or in hardcopy notebooks?
Is the data storage location secured either in a locked fireproof
cabinet for hardcopy or on a server protected by firewall?
Has the data quality been assessed? How many replicates and
how reproducible is the data? Has statistical analysis been
performed on the data? What quality control has been utilized
X
X
X
X
X
X
Both are used
X
Page 14 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 15 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Assessment Criteria for Milestone Completion
by the subcontractor during the data generation and
assessment?
If a protein or peptide has been synthesized, how has the
protein or peptide sequence been verified? What percentage of
the sequences has been randomly verified?
X
If a genetic mutant has been made, how has the mutation been
verified e.g. DNA sequencing, PCR sequence verification?
How stable is the mutation? How has the impact of the genetic
mutation on the bacterial growth been assessed? What is the
sensitivity of the assay?
X
If an aerosol delivery system has been tested, how reproducible
is the delivery to the animal? Have sufficient animal numbers
been tested to determine reproducibility?
X
If UVA/psoralen treatment kills the bacteria but leaves them
metabolically active, how is killing assessed? How sensitive is
the assessment of killing? How is expression of bacterial
epitopes determined?
X
Do UNM and the subcontractor agree that the data supports the
scientific interpretation of the milestone?
Evaluation of Deliverables, as outlined in the
Statement of Work
X
Yes No N/A Comment
Have Standard Operating Protocols have been written by
subcontractor, reviewed by UNM, revised by subcontractor as
requested, and accepted by UNM? The milestone completion
report will not be accepted by UNM until all the SOPs are
accepted by UNM.
X
Has the Milestone Completion Report been written by
subcontractor, reviewed by UNM, revised by subcontractor as
requested, and accepted by UNM?
Has data from the milestone been submitted by the
subcontractor, reviewed by UNM, data presentation revised by
the subcontractor as requested for clarity, and accepted by
UNM?
For deliverable reagents, have the minimum number of vials,
the minimum concentration, the minimum block size and the
minimum weight of tissue been mutually agreed by UNM and
the subcontractor?
X
SOPs used on MS 42 were
submitted and accepted by
UNM and NIAID in
association with MS 40.
Justin Skoble provided 3
PDF files of published
reference also.
X
X
Page 15 of 16
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 42
Institution: Cerus Corporation
Author: Justin Skoble
MS Start: 2/1/07
MS End Date:6/15/08
Report Date: 4/27/09
Version: 2.0
Page 16 of 16
Reviewed by : Barbara Griffith 5/4/09, 5/5/09,
6/26/09, 12/3/09, 1/4/10, 1/5/10, 1/29/10, 2/2/10
Accepted Date: 5/5/09
Assessment Criteria for Milestone Completion
Have bacterial strains and tissues been banked at the
subcontractor’s institution and backup stocks and aliquots been
received by UNM for long term storage? A minimum number
of vials of -20C /-80C bacterial stocks at specified
concentration in glycerol are stored at both institutions. A
minimum size paraffin block or minimum weight of
cryopreserved frozen tissues are stored at both institutions.
Evaluation of SOPs
Do SOPs contain standard sections e.g. purpose, list of
supplies and equipment required including vendors and model
numbers, reagent preparation, method, results expected,
description of data interpretation, criteria for accepting or
rejecting results, description of data storage location, date SOP
is in service, names of people who prepared and reviewed the
SOP?
Can an independent scientist read and understand the standard
operating procedure?
6.3
X
Yes No N/A Comment
X
Relevant SOPs were
already accepted in
association with the MS
40 MSCR.
X
Relevant SOPs were
already accepted in
association with the MS
40 MSCR.
Appendix 3: Additional Data/Figures NOT included in the Text of the
Milestone Completion Report (Section 3 or 4)
6.3.1
Insert
6.3.2
Insert
6.3.3
Insert
Page 16 of 16