Tularemia Vaccine Development Contract: Semi-Annual Technical Report Period: 10/01/2008 to 3/31/2009

Tularemia Vaccine Development Contract: Semi-Annual Technical Report

Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009

Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch

Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel

Contract No.

HHSN266200500040-C

ADB Contract No . N01-AI-50040

Contract Title: Tularemia Vaccine Development Team

Performance Period : 10/1/08 to 3/31/09

Contractor Name: University of New Mexico Health Science Center

Contractor Address: Control ler’s Office MSC-09-5220

1 University of New Mexico

Albuquerque, New Mexico 87131-0001

Authors : C. Rick Lyons, MD, PhD and Barbara B. Griffith, MS

Date of submission: 4/15/2009

Semi-Annual Based on Contributions from ASU, Cerus, LBERI,

Technical Report: UNM and UTSA

Page 1 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Contract No. HHSN266200500040-C

ADB Contract No. N01-AI-50040

Section I: Purpose and Scope of Effort

The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal models and cellular assays vital for testing vaccine efficacy.

Sections II and III: Progress and Planning Presented by Milestone

Active milestones:

2, 5, 7, 8, 9, 10, 11

(UNM &LBERI),

12/13

(UNM &LBERI),

14, 17, 18, 19, 21

(UNM

&LBERI),

29

(UNM &LBERI),

35

(UNM/ASU),

36, 49, 50, 52, 53.

Completed milestones:

1, 3, 4, 6, 16, 25, 26, 27, 28, 32, 33, 34, 39, 40, 43, 48, 51

Inactive milestones:

15, 20, 22, 23, 24, 30, 37, 38, 54

Milestones terminated after initiation:

41, 42, 44, 46, 55, 56, and 57

(MSCR will be written)

Milestones terminated before initiation

:

43

(Cerus)

45, 47, 58, and 59

(MSCR will not be written)

Milestone 2

Milestone description:

Vaccinations performed on relevant personnel

Institution: UNM

/

LRRI

1. Date started: 11/01/2005

2. Date completed: In progress

3. Work performed and progress including data and preliminary conclusions

Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. UNM EOH performed 37 annual health screenings since 8/26/08 for the LVS vaccinees originally vaccinated through April 2008.

Monthly-March 2009: a. Three UNM and possibly 6 LBERI scientists will request vaccinations in 2009. b. USAMRIID tentative vaccination date is June 2009, pending FDA approval

4. Significant decisions made or pending a. 6 month period i. USAMRIID tentatively will resume offering vaccinations to UNM and LBERI in June

2009 if FDA approval is given. ii. UNM (4) and LBERI (33) are vaccinated; UNM and LBERI will offer the LVS vaccinations to 9 more scientists to total up to 46. b. Month of March i. Dr. Lyons received UNM IRB re-approval to allow blood draws on the vaccinated

LBERI and UNM scientists after their LVS vaccinations

5. Problems or concerns and strategies to address a. Nine scientists could be vaccinated in 2009 if USAMRIID receives FDA approval for the new

Tularemia vaccination protocol.

b. USAMRIID may restart LVS vaccinations in June 2009 pending FDA approval c. UNM, True, LBERI, USAMMDA and USAMRIID are extending the CRDA termination date from 6/29/09 to September 29, 2010. The amended CRDA is currently circulating for original signatures required by UNM.

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Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



6. Deliverables completed a. A total of 37 participants (33 LBERI and 4 UNM participants) have received the LVS vaccination since 9/11/07. b. 37 participants have terminated from the USAMRIID SIP, after completing the one year health follow-up at UNM EOH.

7. Quality of performance

Excellent

8. Percentage completed

75% of the scientific work is complete

9. Work plan for upcoming month and next 6 months a. Next 6 months i. Schedule LVS vaccinations for 9 remaining LBERI and UNM scientists in June 2009 b. Month of April i. UNM is obtaining blood donations from LVS vaccinees for immunoassay development and reimbursing participants $40/ donation. ii. UNM will work with 3 UNM and 6 LBERI scientists for the pre-vaccination health screenings required for vaccinations once USAMRIID has FDA approval to offer the

LVS vaccinations again.

Milestone 4


Confirmation of aerosol in vivo in NHP

Institution: LBERI



3. Work performed and progress including data and preliminary conclusions:

Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. Histopathology analysis was completed for Pilot 2 and Pilot 3.

i. The clinical pathology data for pilot 2, as summarized below and are consistent with a response to a chronic bacterial infection.



Lung, TBLN, spleen- lesions are those expected for F. tularensis .



Normocytic, normochromic anemia is compatible with response to chronic inflammatory disease.



Reductions in serum phosphorus and albumin are compatible with sustained anorexia.



Different lesions in TBLNs from the same animal are likely due to their relative positions with respect to lymphatic drainage of the lungs. Suggests caution should be used in interpretation of data from just one lymph node.



Mesenteric lymphadenitis may be consistent with GI uptake of organism, or systemic dissemination to multiple lymph nodes. Other lymph nodes will be examined in future studies.



GI nematode of undetermined significance in one animal. ii. The clinical pathology data for pilot 3 is summarized below.



Lung, TBLN, spleen- lesions are those expected for F. tularensis .



Lung lesions in these animals were smaller and more uniformly distributed

(miliary) than in previous pilot studies, likely due to the greater inhaled dose and earlier death.



Different lesions in TBLNs from the same animal are likely due to their relative positions with respect to lymphatic drainage of the lungs. Suggests caution should be used in interpretation of data from just one lymph node.

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Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009





Nasal cavity lesions in one animal suggest that systemic dissemination from the nasal cavity is possible. a. Work on the Milestone Completion Report was performed.

4. Significant decisions made or pending a. 6 month period- None b. Month of March- None

5. Problems or concerns and strategies to address

None

6. Deliverables completed

LBERI Schu S4 was proven to cause lethality at high doses and SOPs are being written to describe aerosol exposure of NHP.


Good



9. Work plan for upcoming month and next 6 months a. Month of April i. Submit the draft Milestone Completion Report to UNM by April 17, 2009 b. Next 6 months i. Finalize the Milestone Completion Report after review by UNM and NIAID.

Milestone 5

Milestone description: Small

species tested for sensitivity to LVS & generation of immunity against a pulmonary challenge of SCHU S4

Institution: UNM


2. Date completed: pending


Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. s.c. vaccination with a small dose of ~100 organisms of LVS protected Fischer 344 rats against i.t. SCHUS4 challenge b. Fischer 344 rats are orders of magnitude more sensitive to i.t. challenge than to s.c. challenge with SCHU S4 c. The MS5 MSCR for the mouse animal model is drafted.

Monthly-March 2009: a. Experiment Ftc71.2 (Notebook 130 pages 31, 34-35, 43, 48-53) i. The purpose of this experiment was to determine the effect of LVS vaccination dose on the resistance of vaccinated rats to i.t. SCHU S4 challenge. This is a repeat of experiment Ftc71.1 which showed that a s.c. vaccination dose as small as 10 3 protected 5 of 6 Fischer 344 rats against an i.t. challenge of 3.3. x 10 3

SCHU S4. ii. In this repeat experiment, Fischer 344 rats (n = 6) were either left unvaccinated as a negative control or vaccinated s.c. with 6.8 x 10 1 , 6.8 x 10 2 , or 6.8 x 10 4

LVS/rat based on the inoculum concentration and then 37 days later challenged i.t. with 8.7 x 10 3 SCHU S4 iii. This experiment not only confirmed the Ftc71.1 results, but also showed that s.c.

LVS vaccination with even a >10-fold lower dose of 68 cfu LVS was protective

Page 4 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel against i.t. SCHU S4 challenge (Fig. 1). As a model for vaccination by acupuncture in humans, this result suggests that very few LVS organisms would be required to induce effective immunity

100

80

60

40

20

0

Unvaccinated

6.8 x 10

1

LVS

6.8 x 10

2

LVS

6.8 x 10

4

LVS

0 5 10 15 20

Days post challenge

Figure 1. Effect of LVS vaccination dose on the resistance induced in Fischer 344 rats. Fischer 344 rats (n= 6) were vaccinated with LVS and challenged 8.7 x 10 3 SCHU S4. The indicated doses were calculated from the inoculum concentration. The rats were monitored daily for weight loss, clinical signs and survival. b. Experiment Ftc85 (Notebook 130 pages 37-39, 44-45) and Experiment Ftc85,1

(Notebook 130, pages 40-42, 46-47) i. Humans and non-human primates are not only highly susceptible to aerosol

SCHU S4 infection but also to intradermal SCHU S4 infection. The purpose of these experiments was to determine the suscep tibility of naïve Fischer 344 rats to s.c. SCHU S4 challenge. The challenged doses used were 10 1 , 10 3 , and 10 5 cfu; for comparison, the i.t. LD

99

is less than 50 cfu.

ii. Fischer 344 rats were much more resistant to s.c. challenge than to i.t. challenge

(Fig. 2). Almost all of the infected rats lost weight following s.c. challenge but only few of them died. There was no obvious correlation between the challenge dose, the mean time to death and the number of deaths per group that died. The LD

50 was not reached with the challenge doses used, but the s.c. LD

50 is greater than

10 5 cfu, at least 4 logs higher than the i.t. LD

50

. This information provides another facet for us to evaluate the Fischer 344 rat as a model for human infection.

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Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



100

80

60

40

20

0

10

1

10

5

SCHU S4

10

3

SCHU S4

SCHU S4

0 5 10 15 20 25

Days post challenge

Figure 2. Susceptibility of Fischer 344 rats to s.c. SCHU S4 infection. Naïve Fischer 344 rats (n = 6) were infected with SCHU S4 and monitored for 21 days for weight loss, clinical signs and survival. The indicated doses were determined by plating the inocula on cystine heart agar plates

4. Significant decisions made or pending

For the 6 month period

No significant decision was made in this 6-month period. We had already made the decision to use the Fischer 344 rat model as an alternative to the mouse model and to characterize it in detail in a previous semi-annual report


None


For the 6 month period

Determined vaccination dose response in Fischer 344 rats

Determined the sensitivity of Fischer 344 rats to s.c. SCHU S4 challenge


Good


99%

9. Work plan for upcoming month

For Next one month



The only uncompleted work left on this milestone is the analyses of tissues from mice, rats, and NHPs infected with SCHU S4. The tissues have been collected and stained and are waiting to be analyzed by the veterinary pathologist



Complete milestone completion reports for the mouse, rat, and guinea pigs

For Next 6 months



Same as the Next one month plan

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Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Milestone 7


SCHU S4 ED50 in primates determined from selection of challenge dosing

Institution: LBERI


2. Date completed: In progress.


Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. Wave 1 high dose challenges were performed with target doses of 1,000, 10,000, and

100,000. The animals were presented with 1380-1,260,000 CFUs.



All animals died within 3 to 9 days post-exposure.



In general, temperatures decreased dramatically as death approached.



Respiration rates increased to greater than 50% above the average respiration rate for every animal and remained at or near that level until death.



Bacteremia was observed for most animals.



White blood cells increased by day 2 and dropped by day 4, due predominantly to neutrophils. This increase is a relatively common response to infectious diseases.



Large amounts of bacteria were found in spleen, liver, lung, tracheobronchial lymph node

(TBLN), and in all cases but 2, in mesenteric lymph nodes (MLN).

b. Wave 2 low dose challenges were performed with target doses of 25 and 250 CFUs. The animals were presented with 1-2 and 19-90 CFUs.



The majority of animals died between days 8-20. Death did not appear to be dependent on dose. One animal (2 cfu) survived until day 46 upon which it was euthanized. All

NHP were clinically ill.



A few animals had sporadically elevated temperatures; hypothermia was often observed prior to death.

 Respiratory rates rarely increased to greater than 30% of each animal’s average respiratory rate, and those that did demonstrate an increase did so transiently.



Bacteremia was observed for every animal. Duration and amount of bacteria found in the blood were not dose specific.



Hematology showed an increase in white blood cells by day 4 (delayed from what was observed with higher doses in wave 1), that was predominantly due to neutrophils.



High numbers of bacteria were found in the lungs and TBLNs of all animals except the lone survivor. More variable amounts of bacteria were quantified in the liver, spleen and

MLN (again, except for the survivor). Tularemia was found in the eyes of 3 NHP and did not appear to be a dose-dependent phenomenon. Tularemia was found only in the TBLN of the lone survivor and was present at low concentrations (324 CFU/g). c. Wave 3 low doses were performed with target doses of 250 and 500 CFUs. The animals were presented with 237-444 and 624-1150 CFUs.



All animals succumbed between days 4 and 8. The animals that received higher doses died slightly earlier than those with lower presented doses.



Hypothermia was again observed as death approached.



Respiratory rates, like those in the first wave, increased to greater than 50% of the average normal rate and was sustained until death.



Bacteremia was observed on days 4 and/or 6 for all animals. Time to demonstration of bacteremia and bacterial load did not appear to be dose-dependent but rather animal dependent.



Hematology parameters were similar to those observed in wave one with white blood cells increasing by day 2 and due to an increase in neutrophils.

Page 7 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009





High levels of bacteria were observed in the lung, TBLN, MLN, liver and spleen, with the exception of animal 28585 (counts in the MLN below level of detection).

d. In summary, respiratory symptoms in all waves included gasping, coughing, and nasal discharge. These symptoms, however, were not observed in all animals. Other signs and symptoms included skin lesions, ocular tularemia, and hypothermia in advance of death. The

ED50 was determined to be 1-2 cfu.

Monthly-March 2009: a. Histology slide preparation was completed.

4. Significant decisions made or pending a. 6 month period i. Based on the results of Wave 1 and Wave 2, Wave 3 presented target dose was 250 and

500 CFUs. ii. The SCHU S4 ED

50

is 1-2 in cynomologus macaques iii. The chosen challenge dose for the LVS vaccinated challenges (Milestone 8) was 500

CFU. b. Month of March- none.


None


LD

50 is 1.26 CFU of SCHU S4. The LD

99

is 4.23 CFU.


Good.


90% of the scientific work is complete.

9. Work plan for upcoming month and next 6 months a. Month of April i. Respiratory rates and temperatures previously reported will be modified as per the format provided by Kristin DeBord. ii. Histopathology will continue to be analyzed. A draft pathology report will be prepared by the end of April. b. Next 6 months i. The final pathology report will be completed by the end of May. ii. The Milestone Completion report will be written by June 15, 2009 and finalized.

Milestone 8


LVS vaccination protection of aerosol Schu S4 validated in primates

Institution: LBERI


2. Date completed: In progress.


Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. In October 2008, 5 animals were vaccinated with LVS by two routes to later assess protective efficacy against SCHU S4 aerosol challenge. Three of the animals were vaccinated via scarification and 2 via subcutaneous injection to determine if the animals mounted an immune response to LVS. The target vaccination dose was 60 x 10 6 LVS organisms. Blood draws were performed on Day 0, 7, 14, 21, 28, and 35 and PBMCs were prepared for assessment of cellular immunity. Plasma was tested for IgG anti-LVS antibody content. i. Although the target LVS vaccination dose was 6 x 10 7 organisms (calculated to mimic the USAMRIID vaccination protocol), the actual inoculation dose was 2.6 x 10 5 . The

Page 8 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel dose is delivered directly from a vial of Lot 16 LVS (DVC) which was resuspended in

1.4 ml water. LBERI chose this protocol to mimic the USAMRIID protocol. However,

LBERI has repeatedly seen lower titers in the DVC LVS vials than originally reported by DVC. Nevertheless, in order to mimic the USAMRIID protocol, this apparent decrease in titer was purposefully not taken into account as USAMRIID does not titer each vial of vaccine but rather relies on the original titer stated by DVC. ii. Each vaccinated NHP mounted a humoral (IgG antiLVS) and cellular (IFNγ secretion as measured by ELISPOT and proliferation when challenged ex vivo with HK or FF

LVS) immune response. iii. No significant difference in responsiveness to HK or FF LVS antigens was observed when comparing the cellular or humoral immune response of the NHPs immunized by scarification or s.c. injection. This was important as this experiment was set up to test whether we could vaccinate by scarification in preparation for a future experiment in which we would use scarification as the route of inoculation (USAMMDA IND 157 vs. DVC Lot 16 test). b. In January 2009, 6 animals were vaccinated with LVS (3 via scarification and 3 via subcutaneous injection). These animals were vaccinated by two routes to compare the immune response elicited by each route and the ability of the two vaccination routes to protect against a SCHU S4 challenge. The vaccination dose was determined to be 3.1 x 10 4

CFU LVS even though the target dose was 6 x 10 7 CFU. As discussed above (3.a.i.), LBERI assumed that each vial contained 1.4 x 10 9 CFU. LBERI resuspended the vial in 1.4 ml water which should have resulted in a concentration of 1 x 10 9 /ml. The USAMRIID SIP protocol details that LVS is delivered at 1 x 10 9 /ml in a 60 microliter inoculation volume (resulting in 6 x

107 LVS CFU delivered). LBERI attempted to replicate this protocol but when the inoculums was plated, it was determined that the dose contained 0.52 x 10 6 /ml rather than the expected

1 x 10 9 /ml resulting in a delivered dose of 3.1 x 10 4 in 60 microliters. c. In February 2009, 12 NHPS (3 non-LVS vaccinated controls; 6 vaccinated with LVS in

January 2009; 3 vaccinated with LVS in November 2006 (2 with 1.5 x 10 7 LVS by intradermal inoculation and 1 with 2.7 x 10 6 by s.c. inoculation) were challenged with 27-1780 CFUs

(target dose was 500 CFU SCHU S4). i. 9/12 NHPs succumbed to SCHU S4 challenge between days 4 – 16. ii. As with the ED50 study (MS7), hypothermia was a common event prior to death.

Respiration rates increased 30-50% over each anim al’s average, but unlike in the

ED50 study, few animals exhibited sustained increase over 50%. Tissue bacterial burden was more variable than observed previously. Bacteremia was present in all animals except for 3 (1 control animal and 2 animals that were scarified in January

[A03152, 28643, 28671]). Hematology data was similar to waves 1 and 3 of the

ED50 study where total white blood cell counts increased by day 2 due predominantly to an increase in neutrophils (observed in all animals). iii. 3/12 NHPs survived until day 20 (one NHP vaccinated by the i.d. route in 11/06) or

21 (one non-LVS vaccinated control and one inoculated with LVS by scarification in

January 2009) when these three NHPs were euthanized. iv. There were not enough animals to determine whether a significant delay to death was induced by vaccination, however, a trend existed in that the NHPs inoculated by the s.c. route in January 2009 were the last 3 of 9 to succumb (days 10, 12 and 16 post-aerosol challenge). v. All LVS-vaccinated NHPs had increased plasma levels of IgG anti-LVS compared to levels tested prior to vaccination. vi. The cellular immune response of the NHPs vaccinated with LVS in January 2009 was sub-optimal in that only 1 of 3 NHPs vaccinated via scarification and 2 of 3 vaccinated via s.c. inoculation secreted more IFNγ in response to HK LVS postvaccination (day 25) as compared to pre-vaccination.

Page 9 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel vii. This sub-optimal cellular immune response to LVS may be due to the low vaccination dose (3.1 x 10 4 ).

Monthly-March 2009: a. Survivors from the vaccine challenge study were euthanized on approximately day 21 postchallenge. Tissues were taken for pathology, microbiology, and immunologic assessment. b. The humoral immune response (IgG anti-LVS) will be measured on filtered plasma in the

BSL2 laboratory if it can be shown that filtration removes any SCHU S4 bacteria. This testing is currently underway (April 2009). If SCHU S4 cannot be removed, the ELISA will be conducted in the ABSL3 laboratory. c. The IFNγ ELISPOT plate was set up on March 5 th and developed on March 6 th . The plate remains in the ABSL3 until testing can be completed on the ability of VHP (vaporized hydrogen peroxide) treatment to remove any viable SCHU S4. Testing of VHP efficiency on

SCHU S4 spiked plates is currently underway.

4. Significant decisions made or pending a. 6 month period i. Based on data from Milestone 7 vaccinated animals were initially challenged with a target dose of 500 CFU SCHU S4. ii. Based on the failure to reproducibly recover expected numbers of LVS organisms from the vials containing lyophilized LVS provided by DVC (Lot 16), LBERI personnel are re-testing various ways of reconstituting and growing the organism. b. Month of March i. Based on the February 2009 vaccine/challenge study results and in consultation with

NIAID and UNM, a new study was proposed to test four different LVS vaccination doses (10 2 , 10 4 , 10 6 , and 10 8 ) delivered by subcutaneous injection followed by an aerosol challenge of 1000 CFU. ii. Upon further consideration and re-examination of the data showing poor LVS recovery from the lyophilized pellets, it was determined that we could not deliver 10 8 organisms, likely being unable to achieve a concentration of 10 8 /0.12 ml = 8.3 x

10 7 /ml. We therefore proposed to deliver doses of 10 7 , 10 5 , and 10 3 LVS. Our current tests of resuspension of the lyophilized vials of LVS and subsequent determination of vial titer will determine whether we can deliver these doses reproducibly. iii. We await a decision from NIAID personnel on the route of vaccination (i.d. vs. scarification). iv. High titers of IgG anti-LVS antibody do not protect NHPs from SCHU S4 aerosol challenge when delivered at levels of 27 – 1780 ED

50 s. v. Based on the fact that high IgG anti-LVS titers do not correlate with protection against

SCHU S4 aerosol challenge, we have decided to cease excluding NHPs from studies that have high IgG anti-LVS titers. However, plasma titers and PBMC responses to

LVS and SCHU S4 antigens will still be measured on all NHPs entering studies at

LRRI where it is expected that immunological parameters are relevant to the study

(e.g. to serve as a baseline value for any vaccination study).

5. Problems or concerns and strategies to address a. Poor reproducibility of LVS reconstitution and growth from the lyophilized vials received from

DVC (Lot 16). LBERI is consistently finding titers at least 100 fold less than reported by DVC.

Different reconstitution (0.25 ml of water rather than 1 – 1.4 ml), dilution into saline, and growth methods (4 – 5 days on CHAB agar vs. 2 days on Chocolate or CHAB agar) are currently being tested to determine the effect on the LVS titer. b. A data entry error in SCHU S4 preparation was made leading to a dilution error that resulted in targeting 50 rather than 500 CFU in the aerosol delivered in 2/09. More care and oversight needs to be taken to ensure this does not happen in future experiments. A second

Page 10 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel independent scientist will calculate the doses and compare the calculations to the microbiologist’s calculations. c. The first LVS vaccination/SCHU S4 challenge experiment did not show LVS lot16 vaccine protection against a range of SCHU S4 challenges. A repeat LVS vaccination/SCHU S4 challenge experiment is being planned as described under “significant decisions” in section 4 above.


None


Good



9. Work plan for upcoming month and the next 6 months a. Month of April i.

Determine the amount of LVS that LBERI personnel can reproducibly recover from the lyophilized vials of DVC Lot 16 ii.

Consult with UNM and NIAID, to finalize the study design of the LVS vaccination dose/SCHU S4 challenge experiment iii.

Write the Study Protocol and submit to NIAID personnel for review and comment iv.

Analyze the cellular and humoral immune response of the three survivors of the

SCHU S4 challenge on 2/12 and 2/13 by retrieving the ELISPOT plate and plasma from the ABSL3 or conducting the assays in the ABSL3 facility v.

Is histopathology pending on NHP from the first vaccination/challenge experiment? b. Next 6 months i. Perform LVS vaccination dose study followed by an aerosol challenge of 1000 SCHU

S4 CFU.

Milestone 9


Aerosol SOP developed for GLP transition

Institution: LBERI




Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. Pre-qualification data were initially compiled and organized to define acceptable SCHU S4 bioaerosol performance criteria based on a target presented dose of 500 CFU in 3.5 L inhaled. b. A draft SCHU S4 bioaerosol Qualification Plan was submitted to LBERI QA. Following review, minor revisions were required. c. Two days of mock-qualification bioaerosols were performed to evaluate the feasibility of the defined performance criteria. Results demonstrated that, while very close, target aerosol concentrations from two of five runs were outside of the range of acceptable values for each day of testing. It was concluded that the defined criteria (with respect to bacterial aerosol concentration) were too narrow.

Month-March 2009: a. UNM, LBERI, and NIAID decided to increase the target presented dose from 500 to 1000

CFU for GLP transition. This was decided in order to increase the chances of achieving the target dose.

b. No new laboratory work was performed in March 2009

Page 11 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



4. Significant decisions made or pending a. 6 month period i. Mock qualification bioaerosols in conjunction with actual NHP exposures have demonstrated the difficulty in consistently achieving a presented dose of 500 CFU. A broader acceptable challenge target of 1000 CFU was established. b. Month of March- It has yet to be established what the acceptable range will be. A decision is pending as to how much higher and lower than 1000 CFU presented will still be considered acceptable. This will depend significantly on the results from the natural history study.


As stated in Item 4, LBERI needs to determine what will be considered an acceptable range of delivered SCHU S4 in the aerosol (keeping in mind that pulmonary disease is established at or above

89 CFU).


None


Good



9. Work plan for upcoming month and the next 6 months a. Month of April i. Establish a broader acceptable challenge dose range (with input from the UNM

TVDC team and NIAID) and conduct an additional day of mock-qualification runs. ii. Revise and complete qualification plan for the aerosol and submit to UNM for review.

New data from April 2009 mock qualification runs and NHP exposures will be incorporated into the pre-qualification data. This will alter (i.e., improve) the acceptable criteria (e.g., spray factors, pre- versus post-bioaerosol values, etc.) and change the current draft version. All target challenge dose values will change since the challenge dose has been revised from 500 to 1000 CFU presented. b. Next 6 months a. Finalize the Qualification Plan and conduct qualification bioaerosol runs. b. Initiate milestone completion report c. Prepare final deliverable SOP(s).

Milestone 10


Efficacy testing of vaccine candidates (LBERI) and Characterization of selected small animal model (UNM)

Institution: LBERI /

UNM




Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. Animals (19 NHP) were ordered for the testing of the USAMMDA IND 157 LVS vaccine as a test vaccine candidate

Month-March 2009: a. No work was performed on this Milestone during the month of March.

4. Significant decisions made or pending a. 6 month period

Page 12 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel i. The testing of the USAMMDA IND 157 vaccine will be delayed until LBERI further tests the vaccine efficacy of the DVC LVS lot 16 used on the TVDC to date. b. Month of March i. It was decided to reserve the 19 NHPs that arrived 4/1/09 for use in the LVS vaccination dose study outlined and discussed above under Milestone 8.


None


None


Good


2%

9. Work plan for upcoming month and next 6 months a. Month of April: no work planned b. Next 6 months: a. to start the experiment comparing USAMMDA IND 157 and DVC Lot 16, which has been delayed. It is possible that LBERI will order NHPs for the USAMMDA study to arrive on June 1 should we anticipate knowing the results of the LVS vaccination dose study by the end of July. The decision to order monkeys will be made in consultation with Dr. Lyons and NIAID. Next 6 months b. Test the efficacy of USAMMDA IND 157 vaccine vs. DVC Lot 16.

Milestone 11


In vivo GLP NHP model efficacy SOP and efficacy testing of vaccine candidates

Institution: LBERI




Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. The IACUC protocol for the Natural History Study was prepared and approved by the IACUC committee. b. The ES&H work review for the Natural History Study was prepared and approved. c. NHP were ordered and received. d. LBERI completed immunological screening of the NHP to determine IgG anti-LVS titers and the performance of PB MCs in the IFNγ and proliferation assays. i.

None of the 22 NHPs tested had elevated IgG anti-LVS titers. ii.

Analysis of the IFNγ production has not been completed due to technical problems with the ELISPOT reader at UNM. As the plates are dried, they will be read when the reader has been repaired with no adverse effect on the spots. iii.

Proliferative response to HK or FF LVS or SCHU S4 was occasionally observed but was never above 250,000 RLU, and rarely above 150,000 RLU, and thus considered to be background levels

Month-March 2009: a. Natural History Study protocol was prepared and approved. b. NHP (cynomologus macaques) received a physical examination, pole/collar/chair training and were moved into the ABSL3. Pre-exposure temperatures, respiration rates, and biscuit intake as well as behavior were monitored.

Page 13 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



4. Significant decisions made or pending a. 6 month period i. NHP (n=16) will be exposed to 1000 CFU target dose via aerosol (split gender). Four

NHP (2 M/2 F) will be sacrificed on Study Days 2, 4, 5, 6. These animals will be monitored for clinical signs and symptoms, and will have full necropsy. Blood for clinical chemistry, hematology and quantitative bacteriology will be taken daily. Sera will be set aside for determination of cytokine levels. A variety of tissues will be examined histological. Should animals become moribund prior to their scheduled sacrifice, they will be euthanized and blood (if possible) and tissues taken at necropsy.

b. Month of March i. The challenge dose was changed from 500 CFU to 1000 CFU.


For the telemetered arm of the Natural History Study, NHP need to be larger and physically weigh more to accommodate the telemeters, thus animals greater than 2.5 kg were requested. Availability of these larger animals is being investigated in conjunction with orders. These NHP will likely have to be between approximately 2 and 5 years of age.


None


Good



9. Work plan for upcoming month and next 6 months a. Month of April i. Challenges will be performed on April 1, 2009 and April 2, 2009. b. Next 6 months i. NHP for the telemetered portion of the study have been ordered and are set to arrive in June. ii. Data interpretations for the untelemetered portion of the study will continue.

Milestone 11

Milestone description: In vivo GLP model efficacy SOPS developed in one small species and primate and efficacy testing of vaccine candidates

Institution: UNM




Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. Passive immunization with LVS immune rat serum protected naïve rats against i.t. SCHU

S4 challenge. The level of protection was limited compared to s.c. LVS vaccination; rats immunized with 250

 l immune serum were protected against a maximum challenge dose of ~700 cfu SCHU S4. When the volume of immune serum transferred was reduced, the level of resistance decreased accordingly. b. Preliminary studies indicated that the IgG component in immune rat serum is involved in protection against SCHU S4 challenge c. Initial bacterial SCHU S4 replication in the lungs of passively immunized rats was the same as naive rats but systemic replication in the liver and spleen was intermediate

Page 14 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel between naïve and LVS vaccinated rats. Passively immunized rats cleared SCHU S4 infection with a delayed kinetics compared to LVS vaccinated rats d. Infected tissues from passively immunized rats will be compared with LVS vaccinated rats, naïve rats, and rats that received normal rat serum

Monthly-March 2009: a. Experiment CDep4 (Notebook 141, pages 16-22) i. The purpose of this experiment was to optimize protocol for depleting Fischer 344 rats of CD4 T cells. T cells will be depleted to determine their role in the cellular immune response for protection against SCHU S4 challenge. ii. Based on the results from experiment CDep3, we treated naïve rats i.p. with 100, 250 and 500

 g of OX-38 ascites fluid (depleting antibody for CD4 T cells). iii. Spleens of normal rats contain about 25% CD4 T cells. This is what we observed when the rats were treated with the isotype control 55-6 ascites fluid (Fig. 3) iv. When the rats were treated with 100

 g OX-38 ascites fluid, the number of CD4positive T cells in the spleen was reduced by 10 percent; there was no advantage to using 250

 g ascites fluid relative to the results with 100ug. When treated with 500

 g, the CD3 expression on CD4-positive T cells disappeared. Since there was no

CD3 staining on CD8-positive T cells either, we think we may have left out the CD3 staining antibody in the experiment. We do not know if this level of depletion will be sufficient to obtain a detectable effect; if not, we will use a cocktail of depleting (OX-

38) and inactivating (W3/25) ascites fluid.

Figure 3. Titration of ascites fluid for depleting CD4 T cells from Fischer 344 rats. Fischer 344 rats were treated i.p. with the indicated amount of 55-6 (isotype control) and OX-38 (anti-CD4) ascites fluid. 1 week after treatment, the spleens were removed and analyzed for CD4 and CD8 T cells by flow cytometry. b. Experiment Ptran12C (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and Results\Gopi's experiments\Ptran\Ptran-12C) i. The purpose of this experiment was to confirm the growth kinetics for SCHU S4 in passively immunized rats. UNM is exploring the role of the humoral immune response in protection to SCHU S4 challenge.

Page 15 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel ii. In Ptran-12B, we observed a second peak in the bacterial burden 10 days after infection. This could be the point in the infection when the transferred immune serum lost its protective effects. We wanted to repeat this experiment in Ptran-12C and focus on the time points leading up to day 10 post-infection. iii. Naïve rats were treated with 250  l immune rat serum and challenged 1 day later with 330 SCHU S4. On days 5, 7 and 10 p.i., 3-4 rats were euthanized to measure tissue bacterial burden. iv. The results of this experiment are similar to Ptran12B (Fig. 4). It now appears that there may not be a second peak in the bacterial burden; instead, the bacterial burden actually continued to increase until day 10 of infection when it started to decline. The

3 experiments of this series have elucidated the kinetics of SCHU S4 proliferation in passively immunized rats compared to LVS vaccinated and naïve rats and have enabled us to compare the effect of passive immunization to active LVS vaccination

Lung Liver Spleen

10 10 10

8 8 8

Ptran 12B

Ptran 12C

6 6

6

4 4

4

2 2

2

0 3 6 9 12 15

Days Post-challenge

18 21 0 3 6 9 12 15


18 21 0 3 6 9 12 15


18 21

Figure 4.

Kinetics of SCHU S4 growth in passively immunized Fischer 344 rats.

Naïve rats (n = 3 or 4) were treated i.p. with and 10 p.i., the infected rats were euthanized to measure tissue bacterial burden. For comparison, the data from

Ptran12C (Δ) was overlaid on the data from Ptran12B (■) c. Experiment PtrIgG-1(L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and

Results\Gopi's experiments\PtrIgG) i. The purpose of this experiment was to show that IgG is a protective component in the immune rat serum that protects rats against i.t. SCHU S4 challenge. ii. IgG was purified from immune rat serum by ammonium precipitation and Melon-Gel

(Pierce) purification. The titer for the resulting purified product was compared to the anti-LVS titer in immune rat serum by ELISA. Based on the anti-LVS titer, 3 times more (by mass) purified IgG was passively transferred into rats than the unpurified immune serum to ensure that any protective effect can be observed. iii. The results showed that purified IgG protected rats against i.t. challenge with 330

SCHU S4, suggesting that IgG against F. tularensis is a protective factor contained in the immune serum (Fig. 5). We cannot rule out the participation of other factors in the immune serum

Page 16 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



100

80

60

40

20

0

Naive (0.25ml PBS)

IRS (0.25ml)

Immune IgG

0 5 10 15 20

Days P.I.

Figure 5. Fischer 344 rats (n =5) were injected i.p. with PBS, LVS immune serum or IgG purified from LVS immune serum. The amount of purified IgG transferred was 3 times more than the amount of anti-LVS IgG contained in 0.25 ml immune rat serum. One day after treatment, the rats were challenged i.t. with 330 SCHU S4. The infected rats were monitored daily for survival. d. Experiment Phist-1 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and

Results\Gopi's experiments\phist-1) i. The purpose of this experiment was to compare the tissues from rats that were left untreated, treated with normal or LVS immune rat sera, or vaccinated with

LVS and then challenged i.t. with SCHU S4. This experiment may provide some insight into the protective mechanism of passive immunization with immune rat serum ii. Rats were either vaccinated s.c. with LVS one month before SCHU S4 challenge or injected i.p. with 150

 l PBS (Naïve), normal rat serum (NRS) or immune rat serum (IRS) one day before SCHU S4 challenge. iii. 3 rats from each group were euthanized throughout the course of infection to collect the lungs, lung draining lymph node, liver and spleen. iv. Slides of these tissues were processed and stained by an external commercial vendor.

The veterinary pathologist has reviewed the slides and will provide a summary of results by the next reporting period.


For the 6 month period -- We decided it was essential to purify IgG from LVS immune rat serum to show that protection was truly mediated by IgG Our preliminary results suggest that IgG is a component of the protective immune response but does not rule out non-immune protective components.


None


For the 6 month period -- Showed that humoral immunity can play a role in controlling low dose respiratory SCHU S4 infection


Good


40%

Page 17 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



9. Work plan for upcoming month a. For Next one month i. Determine whether CD4 and/or CD8 T cells are required to protect LVSvaccinated rats against i.t. SCHU S4 challenge ii. Purify IgG from normal and immune rat sera for repeat of PtrIgG-1 b. For Next 6 months i. Characterize the contribution of humoral and cell-mediated immunity to control of respiratory SCHU S4 infection and the interaction between the two. We will examine the role of B cells in the protection of LVS vaccinated rats and the role of T cells in the passively immunized rats. ii. Determine whether immune mouse serum (Experiment Pmouse1) and human convalescent sera from Martha’s Vineyard protects rats against i.t. SCHU S4 challenge

Milestone 12/13


Assays for detecting relevant immune responses in animals & humans developed and compared to those in other species.

Institution: LBERI

1.

Date started: 2/23/2006

2.

Date completed: In progress

3.

Work performed and progress including data and preliminary conclusions

Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. LBERI observed that some non-LVS va ccinated animals respond to FF LVS by IFNγ secretion and that occasionally there are high backgrounds in the IFNγ ELISPOT assay. It was unknown if these responses would remain consistent if animals were bled on separate days or whether the responsiveness was inconsistent from day to day. To determine which was the case, PBMCs isolated from non-LVS vaccinated animals were re-analyzed in the

IFNγ ELISPOT assay. i. The responsiveness to FF LVS was reproducible; however, the high background response was not, likely being due to variable platelet/RBC contamination of the

PBMCs with each blood draw. b. LBERI tested the ability of previously frozen and thawed PBMCs to produce IFNγ as measured by the IFNγ ELISPOT assay. Two different protocols were tested (Cerus and

CTL). i. Based on relatively equivalent performance of the thawed PBMCs in the assay (both protocols resulted in variable recovery of the activity seen in fresh cells), and the relative ease of the Cerus protocol as compared to the CTL protocol, LBERI chose th e Cerus protocol to use moving forward. UNM and NIAID supported LBERI’s choice of the Cerus protocol. c. Since it had been observed that some non-LVS vaccinated animals respond to formalin-fixed and heatkilled LVS and SCHU S4 antigens in the IFNγ ELISPOT assay, mutant LVS and

SCHU S4 organisms that lack the O-antigen (LPS) were obtained from Dr. Anders Sjost edt’s lab. These heat killed and formalin fixed mutants were tested in the IFNγ ELISPOT assay. i. There appeared to be differences in the response to the O-mutant antigens in the

IFNγ ELISPOT assay but when protein content of each antigen preparation was tested by BCA to determine if equivalent antigen doses were being delivered to the wells, it proved to be untrue. The data showed that most of the antigen preparations were not equivalent in protein content and previous data needs to be re-analyzed.

Page 18 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Month-March 2009: a. Discussions with Dr. Lyons’ laboratory personnel regarding how to proceed with testing of the protein content of the LVS antigen preparations resulted in a protocol which both Dr. Lyons’ and Dr. Wilder’s personnel will follow. i. LVS will be grown in Chamberlain’s broth for 24 hours. ii. Dilutions of the LVS will be made and plated for growth and also lysed to determine the protein content at each dilution. The lysis buffer is one that was chosen from a literature search and was used by Dr. Anders Sjostedt personnel. Protein analysis of the lysates will be performed using a kit (Pierce) that will not be affected by the 1%

SDS or 20% 2-ME in the lysis buffer. iii. LVS will also be fixed and heatkilled according to Lyons’ laboratory protocols at both

UNM and LRRI. Aliquots will be lysed and protein content will be plotted on the

CFU:protein content curve determined using the live LVS to determine the CFU equivalents in each. b. A pool of plasma containing IgG anti-LVS was prepared from nine LVS-vaccinated NHPs and its performance tested in the IgG anti-LVS ELISA. Two independent assays were run

(2/23/09 and 3/18/09). See Figure 1. i. Aliquots were frozen and will be used in all upcoming IgG anti-LVS ELISAs to prepare a standard curve with arbitrary units assigned. We propose designating that the pooled plasma contains 100,000 Units/ml such that the resultant standard curve would look as presented in Figure 1B.

A

Page 19 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



B

Figure 1: Two independent runs of a pooled positive control reference standard for use in future IgG anti-

LVS ELISAs. Panel A shows the data plotted against dilution factor. Panel B shows the data with the assumption that the pooled plasma contains 100,000 Units IgG anti-LVS/ml.

Data storage: Raw Data \\Saturn\Group\Wilder Lab\TVDC\LVS ELISA\LVS ELISA DATA

STATVIEW\ 022309 30’.xls and ...03182009

30 xls.; and in the TVDC 6 (9616) bound notebook pages 86

– 87 and in the TVDC 7 (9633) bound notebook pages 8 – 9

4. Significant decisions made or pending a. 6 month period i. LBERI chose the Cerus freeze/thaw protocol for NHP cells stored on the TVDC contract. b. Month of March i. A positive control NHP plasma sample from pooled vaccinated NHPs was prepared for use in the IgG anti-LVS ELISA.

5. Problems or concerns and strategies to address a. LBERI needs to standardize quantitation of the LVS and SCHU S4 antigen preparations

(protein content versus CFU/mL). The plan is to construct a standard curve correlating

CFU/ml and protein content using LVS and SCHU S4; aliquots will be plated and lysed; lysates will be measured for protein content. Preparations of heat-killed and formalin-fixed

LVS will also be lysed and measured for protein content; the standard curve will allow correlation to CFU/mL.


None


Good



9. Work plan for upcoming month and next 6 months a. Month of April

Page 20 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel i. Determine the relationship between LVS protein content and CFU/mL. b. Next 6 months i. Reanalyze the prior IFNγ ELISPOT assay data based on the new determination of LVS protein content in the antigen preparations ii. Repeat the analysis of protein content and CFU/ml for SCHU S4 antigen preparations. iii. Write the MSCR.

Milestone 12/13-UNM


Assays for detecting relevant immune responses in animals & humans developed and Compare assays in animal models (sensitivity)

Institution: UNM

1. Date started:

7/15/06 (MS12) and 12/06 (MS13)

2. Date completed: Pending


Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. We developed and optimized an ELISA to measure the titer of anti-Ft antibodies in LVS vaccinated humans b. We have also been testing an IFN



ELISpot assay from Mabtech for detection of Ftspecific cellular responses in humans?

Monthly-March 2009: a. Experiment Ftc80.1 (Notebook 128, pages 51-54) i. The purpose of this experiment was to test the Mabtech human IFN



ELISpot kit.

We used fresh PBMC from a LVS vaccinated human volunteer and an unvaccinated control human volunteer from UNM. 2 x 10 5 PBMC and increasing concentrations of heat-killed LVS antigen preparations were used in the ELISpot assay. ii. We detected IFN



production from cells not only from the vaccinated volunteer but also from the unvaccinated control volunteer. We suspect that this control volunteer has some baseline response to F. tularensis and that PBMC isolated from other control volunteers or from blood from the local blood bank would be completely negative.


For the 6 month period -- We realized during this 6-month period that we cannot normalize the responses in our assays because the protein/antigen content in the various heat-killed and formalin fixed bacterial preparations may be different. To correct this problem, we have to correlate bacteria cfu to the protein concentration


None




For the 6 month period -- We developed an ELISA for measuring FT-specific antibody titer in vaccinated humans


Good


72%

Page 21 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



9. Work plan for upcoming month a. For Next one month i. Isolate and test PBMC from buffy coats purchased from the local blood bank in

Mabtech’s human IFN 

ELISpot assay. These will be used as a source of unvaccinated cells in the future. ii. Establish a standard curve correlating the total protein in an antigen preparation to the number of bacteria used to make the antigen preparation b. For Next 6 months i. Isolate and test PBMC from LVS vaccinated volunteers at UNM and LBERI to determine the detection limit of the ELISpot assay, using PBMC from the local blood bank as a negative control ii. Develop the micro-agglutination titer for vaccinated individuals

Milestone 14


Assays in vaccinated humans validated (sensitivity)

Institution: UNM

/LBERI


2. Date completed: in progress


Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY

No work was done during this 6-month period because we are still developing the assays and reagents in Milestone 12/13

Monthly-March 2009:

No work was done

4. Significant decisions made or pending a. For the 6 month period i. We decided to postpone testing sera from convalescent tularemia patients on

Martha’s Vineyard until we have developed a method to normalize the heat killed and formalin-fixed F. tularensis preparations. We also decided to postpone testing PBMC from these Martha’s Vineyard patients until we have determine the sensitivity of the Mabtech human IFN



ELISpot kit


NA


For the 6 month period - none


NA


5%

9. Work plan for upcoming month a. For the 6 month period i. All work is contingent on the availability for assays and reagents described in

Section 4 ii. Determine the titer of anti-Ft antibodies in sera from convalescent tularemia patients on Martha’s Vineyard iii. Quantify the number of IFN



producing cells in PBMCs from convalescent tularemia patients on Martha’s Vineyard

Page 22 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Milestone 17


In vitro assay for analysis of cellular and humoral elements of the immune response in vaccinated human and animal’s response to F. tularensis established

Institution: UNM





No work was done during this 6-month period because we are waiting for human and rat assays to be developed so that we can selectively deplete effector subsets in vitro


No work was done


For the 6 month period - All work initially done under this milestone to characterize the Fischer 344 rat model will be completed under Milestone 11


NA




NA


0%


No work planned

Milestone 18


Role of specific



T cells in protection

Institution: UNM

/LBERI


7/1/08




No work was done during this 6-month period


No work was done




NA

Page 23 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009






NA


5%


No work planned

Milestone 19


Interaction between human alveolar macrophages and F. tularensis

Institution: UNM


12/15/06




We found that human alveolar macrophages can phagocytose SCHU S4 and support bacterial replication, but human alveolar macrophages cannot be activated by the cytokines TNF and IFN



to control bacterial growth

Monthly-March 2009: a. Experiment FT-AH-11(L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and

Results\Andrew's experiments\FT-AH-11), FT-AH-12 (L:\Lyonslab\Tularemia\Tularemia

Contract Folder\Experiments and Results\Andrew's experiments\FT-AH-12), FT-AH-15

(L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and Results\Andrew's experiments\FT-AH-15) i. These 3 experiments are the last in the series of experiments to determine the effects of cytokines TNF and IFN

 on the growth of SCHU S4 in naïve human alveolar macrophages (AM). ii. In two previous experiments, FT-AH-5 and FT-AH-6, we found that cytokine treatment had little, if any, effect on SCHU S4 growth. This suggested that human naïve alveolar macrophages can phagocytose SCHU S4 but cannot control its growth. iii. Because of possible person-to-person variations, we repeated this experiment with 2 additional donors (FT-AH-12 and FT-AH-15). As seen before, we found robust

SCHU S4 proliferation but cytokines had no effect (Fig 6) on the alveolar macrophage ability to control SCHU S4 growth. iv. In FT-AH-11, we used LPS from E. coli strain O125:H7 as an alternative method to activate AM. LPS is a know inducer of TNF and IFN



production. AMs were incubated with 1ug/mL LPS and them infected 48 h later with Schu4 at MOI = 10.

Infected AMs were incubated for another 48 hours before the bacterial burden was determined. This method of AM activation also failed to control SCHU S4 growth (Fig. 7). v. Based on the lack of inhibitory activity in AMs from 5 different donors, we decided to terminate this line of investigation. However one wonders how AM contribute to the pathogenesis and control of pneumonic tularemia

Page 24 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



5

4

3

C on tr ol

IF

N g

TN

Fa

IF

N g

+

TN

Fa

Figure 6. Failure of IFN 6 human alveolar macrophages were incubated with SCHU S4 (MOI = 10) without any cytokine (control) or in the presence of

Fresh cytokines were added at 24 hours post-infection. At 48 hours post-infection, cells were lysed by addition of sodium deoxycholate to 0.1%, and serial lysate dilutions were plated on cystine heart agar.

6

5

4

3

2

- LPS

+ LPS

1

0

3

48

Time post-infection (hours)

Figure 7. Failure of LPS stimulated human alveolar macrophages to limit SCHU S4 growth. 10 6 human alveolar

E. coli strain O125:H7. After 48 hours, the alveolar macrophages were infected with SCHU S4 at MOI of 10 in the presence of additional LPS and incubated for an additional 48 hours. Cell lysates were serially diluted and plated on cystine heart agar. Data points indicate mean +/- standard deviation from duplicate wells


For the 6 month period - Terminate work with human alveolar macrophages if all required AM contract work has been completed


NA


For the 6 month period – characterized the uptake and bacterial replication in alveolar macrophages and the anti-bacteria activities of alveolar macrophages after cytokine stimulation


Good


25%

Page 25 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009




Review contract documents to ensure all proposed objectives in this milestone have been completed.

Milestone 21


Correlates of protection: in vitro assay or other readout of effector function of Ft developed for multiple species.

Institution: LBERI

1.

Date started: 4/8/2008

2.

Date completed: In progress

3.

Work performed and progress including data and preliminary conclusions


No work was performed during this reporting period.

Month-March 2009:

No work was performed during this reporting period.

4. Significant decisions made or pending a. 6 month period: None b. Month of March: None


None


None


Good



9. Work plan for upcoming month and next 6 months a. Month of April i. Repeat the intracellular cytokine staining (ICCS) assay and include a positive mitogen control (Con A). PBMCs from the NHPs vaccinated in October 2008 will be used in the assay. b. Next 6 months i. Continue to work on the ICCS assay to perfect it and determine the timing of cytokine production ( IFNγ, TNFα and IL-2), as well as the cells that are producing them. ii. LBERI will consult with both Amanda Dubois (UNM) and Cecilia Rietz (future employee at

LRRI) who both have expertise in this assay.

Milestone 21


T cell-induced macrophage killing of intracellular bacteria

Institution: UNM


12/15/06



Page 26 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009




Our initial plan was to use human monocyte derived macrophages (MDM) as a target/effector in this correlate of protection assay. However, generating MDM is a very time-consuming process and is not practical for a clinical assay. As an alternative, we tried human PBMC. We showed that human PBMC support vigorous SCHU S4 growth. Pre-activation of PBMC for 48 h with IFN

 and TNF sufficiently activated their effector function to reduce the SCHU S4 burden by 3-4 orders of magnitude, a good range for differentiating vaccinated and unvaccinated individuals. These results suggest that PBMC may be a good alternative to MDM in this assay. Work is in progress to specifically activate PBMC against F. tularensis.


Experiment FT-AH-13 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and

Results\Andrew's experiments\FT-AH-13)



The purpose of this experiment was to repeat the FT-AH-8 which showed that PBMCs have the potential to limit/reduce SCHU S4 growth by almost 4 orders of magnitude. This was an important finding because it suggested that an assay involving PBMC may have the resolution to enable us to distinguish between vaccinated and unvaccinated humans. This resolution is critical to a correlate of protection assay.



In this experiment, we used PBMCs from both LVS vaccinated and unvaccinated human volunteers, although one would not expect that non-specific activators such as TNF and IFN

 to induce differential effects in these two populations. PBMC were pretreated with 100 U/ml

TNF and/or IFN



48 h before SCHU S4 infection at MOI = 1.

 Naïve PBMC did not support SCHU S4 growth in this assay. However, with vaccinated

PBMCs, IFN



reduced the SCHU S4 burden by 3 logs and the combination of IFN



and TNF reduced the burden further by another order of magnitude (Fig. 8). These results also suggest that vaccinated PBMC without any manipulation are unable to control SCHU S4 growth.

6

5

4

3

2

1

0

N ai ve

+

IF

N g

+

TN

Fa

+

IF

N g/

TN

V

Fa ac ci na te d

+

IF

N g

+

TN

Fa

+

IF

N g/

TN

Fa

Figure 8. 2.5 x 10 6 PBMCs from a naïve or vaccinated human volunteer were cultured in the absence of cytokine

Cytokines were replenished every 24 h. After 48 h, PBMCs were infected with SCHU S4 (MOI = 1) and incubated for

Page 27 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel an additional 72 h. The infected PBMC cells were lysed by addition of sodium deoxycholate to 0.1% and serial dilutions were plated on cystine heart agar plates to enumerate the SCHU S4 CFU. Data represent mean +/- standard deviation from at least 4 replicate wells per condition

Experiment FT-AH-9 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and

Results\Andrew's experiments\FT-AH-9)



The purpose of this experiment was to determine whether pre-activation of PBMCs with heatkilled LVS or LPS would lead to control of SCHU S4 growth



In previous experiments, PBMCs were pre-activated with recombinant TNF and/or IFN



to determine whether under optimal conditions human PBMCs are capable of controlling SCHU S4 growth and be used in an assay to distinguish vaccinated and unvaccinated humans.



Having demonstrated that cytokine activated PBMC can control SCHU S4 growth, we wanted to determine whether human PBMCs can be pre-activated specifically with heat-killed LVS to produce similar results



PBMCs from an unvaccinated and a vaccinated human volunteer were pre-incubated with 1ug/ml

LPS ( E. coli strain O127:B8) or heat-killed LVS (HK-LVS) at MOI = 1 for 24 h before SCHU S4 infection and maintained in the presence these factors for 72 h after infection.



Figure 9 suggests that pre-activation with HK-LVS was not sufficient to induce PBMCs to control bacterial burden. This results suggest that restimulation for a longer period of time may be necessary to expand tuli-specific T cells sufficiently for a detectable inhibitory effect

7

6

5

4

3

2

1 unvaccinated PBMC unvaccinated PBMC + LPS unvaccinated PBMC + HK-LVS vaccinated PBMC vaccinated PBMC + LPS vaccinated PBMC + HK-LVS

0

3

72

Time post-infection (hours)

Figure 9. Both vaccinated and unvaccinated human PBMCs pre-stimulated with heat killed LVS failed to control

SCHU S4 growth. 2.5 x 10 6 PBMC from an unvaccinated and a vaccinated human volunteer were stimulated with

1ug/mL E. coli strain O127:B8 LPS or heat-killed LVS (HK-LVS) at MOI of 1. After 24 h, PBMCs were infected with

Schu4 at MOI = 1. At 72 h post-infection, cells were lysed by resuspension in water and serial dilutions of the cell lysates were plated on cystine heart agar to enumerate SCHU S4 CFU. Each bar represents the mean +/- standard deviation from two replicate wells

Experiment Ftc84 (Notebook 128, pages 40-45) and Ftc84,1 (Notebook 128, pages 59-63)



The purpose of these experiments was to develop an assay in the rat similar the one just described for human PBMC.



Approximately 7 ml of whole blood was collected from 2 rats and PBMCs were isolated using

Optiprep density gradient. 2.8 x 10 6 rat PBMC were recovered and used to determine the viability and recovery over a 3-day period. On day 1, the recovery was 78.5% and the viability was 93%; on day 2, the recovery was 77% and the viability was 90.4%; on day 3, the recovery was 66% and the viability was 81%.


For the 6 month period - We will focus on PBMC from humans and rats for further development of this assay


Page 28 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



NA


For the 6 month period -- none


Good


59 %


For the next One month



Determine whether restimulating PBMC from LVS vaccinated human volunteers with heat killed LVS for a longer period of time would result in better control of SCHU S4 growth



Develop and optimize a rat PBMC assay similar to humans



Evaluate the usefulness of LVS/lux operon in RAW 264 murine macrophage-like cell line.

The availability of a light producing LVS or SCHU S4 will shorten the time needed to perform the assay compared to culturing on agar.

For the next 6 months

Determine whether the PBMC assay can differentiate between LVS vaccinated and unvaccinated individuals at UNM, LBERI and the local blood bank

Milestone 26

Milestone description

:

Confirmation of gene and protein expression

(develop HTP SOPs for ORF library production, protein library production, and protein purification)

Description: Prepare a high-throughput protein production system



Select and test ORF expression constructs



Select and test IVT Protocols



Select and test protocols for protein purification

Institution:

ASU-Sykes


3/02/2006

2. Date completed:

1/5/2009


Semi-Annual: 10/1/2008 to 3/31/2009, Summary and Highlights only



Completed new protocol development for ORF production and expression construct assembly



Completed new protocol development for IVT polypeptide production



Completed new protocol development for protein purification



MS 26 completed and drafted milestone completion report

Monthly-March

No new work; Milestone completed


MS26 is completed.


None


IVT library of FTU polypeptides completed and delivered to Terry Wu/Rick Lyons on 1/6/09 on MS28

Page 29 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009




Excellent


100%


N/A

Milestone 28

Milestone description: Generation of polypeptide libraries

(Optimize IVT protein-fragment production, Develop IVT protocol for high-throughput production, Validate immunogenicity of protein-fragments, Full scale production of protein-fragment library, Purification of proteinfragment library, Array protein-fragment into overlapping pools, Ship to UNM)

Milestone description: Build SCHU4 proteome



Build ORF expression library corresponding to proteome (active)



Generate complete protein-fragment library (active)



Array protein-fragments into measurable pools for T cell stimulation (inactive)

Institution:

ASU-Sykes


03-01-2007


Pending





Completed ORF expression library



Completed production and arraying of polypeptide library (2229 Ft polypeptides)



Half of pooled polypeptides were shipped to UNM 1/5/09 and half are stored frozen at

ASU



MS 28 completed and drafted milestone completion report

Monthly-March:



No new work; Milestone completed


MS28 completed


None


IVT library of FTU polypeptides completed and delivered to Terry Wu/Rick Lyons on 1/6/09


Excellent


100%


N/A

Milestone 29

Page 30 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009




Analysis of T cells from lymph nodes & T cell epitopes

.

Institution: LBERI




Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. The IACUC protocol was amended to allow for LVS bronchoscopy to LVS boost the 2 subcutaneously LVS-vaccinated NHPs. The animals were vaccinated in October 2009 under the scope of Milestone 8. b. LVS bronchoscopy of one NHP was performed in February 2009. Necropsy was performed day 12 post-LVS boost. Lymph nodes and splenocytes were transferred to UNM for use in the ASU Ft polypeptide library cellular screening assay. Analysis of PBMC and spleen responses to HK and FF LVS was started.

Month-March 2009: a. The analysis of the PBMC and splenic responses to LVS was not completed due to technical difficulties with the ELISPOT reader.


6 month period- None

Month of March- None


The ELISPOT reader at UNM needs to be fixed. We are currently working with the manufacturer to troubleshoot the problem.

6.

Deliverables completed

None

7.

Quality of performance

Good

8.

Percentage completed


9.

Work plan for upcoming month and next 6 months

Month of April i. Analysis of the PBMC and splenic responses to HK and FF LVS will be completed.

Next 6 months i. The experiment will be repeated when UNM personnel need more vaccinated NHP spleen and LN cells for peptide screening.

Milestone 29


Analysis of T cells from NHP lymph nodes and T cell epitopes

Institution: UNM


10/1/08



Page 31 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Semi-Annual: 10/1/08 to 3/31/09-SUMMARY/HIGHLIGHTS ONLY a. We screened the F. tularensis polypeptide library from ASU with splenocytes and tracheobronchial lymph node cells from a cynomologus macaque that had been vaccinated and boosted with LVS. We identified several positive peptide pools but for most of them the positive results were not confirmed by the duplicate sample or by the second tissue.

Monthly-March 2009: a. no work done

4. Significant decisions made or pending a. For the 6 month period i. We developed the strategy with ASU to screen the polypeptide library ii. We need to decide how to confirm the positive results from the 1 st round


NA.






Good


15%

9. Work plan for upcoming month a. For the Next month - Based on discussions at the ASU site visit, UNM will test individual peptides from a pool that tested positive and a pool that tested negative using frozen splenocytes and lymph node cells b. For the Next 6 months -- test additional peptides if necessary

Milestone 35

Milestone description: Array hybridization with mouse RNA from virulent SCHU S4 infection and RT PCR confirmation of candidates

Institution: UNM/

ASU Johnston


8/1/2006




No new work performed since ASU made no request for RNA samples

Monthly-March 2009: No work done




None




Page 32 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Good


25%

9. Work plan for upcoming month a. For the 6 month period -- we will provide RNA and DNA when requested by ASU a. ASU requested lung RNAs from 2 more mouse and 2 more rat early kinetics of

SCHU S4 infection experiments, using 2000 CFU of SCHU S4 delivered to the lungs.

Milestone 35


Array hybridizations with mouse RNAs from virulent Schu 4 infection &

RT PCR confirmation of candidates.

Institution: UNM/

ASU-Johnston


04-01-2007


Pending





Performed LAPT processing on two mouse and one rat time course experiment. Animals were infected with virulent Francisella tularensis SCHU S4 and lungs harvested at 1,3,5,7,and 24 hours post infection.



Gene expression data were median normalized between experiments from at least two sets of hybridizations per time point and the data were bioinformatically averaged. The expression patterns across the early kinetics time course were use to select the top two hundred genes that were either up-regulated or down-regulated across the time course within a species response.

The data shown in Figure 1 are a heatmap of the mouse time-course samples for both the upregulated and down-regulated gene lists.

Page 33 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Figure 1. Heatmap of the average of the two mouse time courses top two hundred genes that follow an up-regulation or down-regulation of expression across the time course of the infection.

File locations … R:\GeneVac\FTU\Contract\Microarray\Milestones\35\Lapt_Comparisons\LAPT29, 30, 31 tc1 tc2 tc3 comparisons\heatmap.png



Once ASU has gene lists that are identified (see Milestone 36 ongoing), ASU will compare the gene expression profile obtained by microarray analyses with the gene expression detected by quantitative polymerase chain reaction analyses. ASU established a relative quantification qPCR analysis using the MutS gene as the normalization control. The test gene chosen was iglC because of its association with virulence as part of the pathogenicity island. The samples under analysis were from the initial dose response experiment in which mice were challenged with varying doses of SCHU S4 bacteria (10 7 to 10 1 ) and the RNA was isolated from the lungs taken at 4 hours post infection for LAPT. Figure 2 below represents the amplification plot of the Sybr green incorporation during qPCR analyses. The samples in orange hues represent the iglC gene traces and those in the green hues are MutS gene traces; each grouping represents the sample from the highest to lowest bacterial challenge

(10 7 to 10 1 ). The major findings are that the iglC traces generated detectable cycle threshold calculations from each of the doses used in the challenges. This was not observed with the

MutS control gene. The curves of the MutS amplifications were biphasic indicating poor specificity of amplification. Importantly, only the samples from mice challenged with 1,000 organisms or demonstrated reliably detectable traces.

Page 34 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Figure 2. Table on the left represents the Francisella tularensis bacterial loads in mouse lungs at various times after challenge with 1000 CFU bacteria intranasally. The graph on the right represents the qPCR analyses of iglC and MutS gene expression levels in mouse lungs after challenge with various doses (10 7 to 10 1 ) of F. tularensis SCHU S4.

Notebook/File locations …, ASU: Notebook 804, Relative Quantification, page 134.


1000 CFU of SCHU S4 in the mouse lungs is not a sufficiently high dose for reproducible qPCR detection.


The limit of detection in the qPCR analyses for the MutS gene indicates that we need to have samples with higher initial bacterial loads. Since the samples that are currently available have lower bacterial loads, ASU requested that UNM provide new experimental mouse and rat lung RNA samples. UNM will expose the mice and rats intranasally with doses established that result in a minimum of 1-2,000 bacteria being deposited in the lung by 1 hour post infection.


None


Good


80%

9. Work plan for upcoming month/6 months

For the next month:

The replicate samples of the currently prepared LAPT samples from the two mouse and the rat kinetic time course experiments will be hybridized and combined with the previous data analyses.

Page 35 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



For the next four months:

This milestone should be completed within the next 4 months (by 7/30/09). For the completion of this milestone additional animal challenge experiments will be performed at UNM. The key variable will be to deliver approximately 2,000 organisms to the lungs for the early time point measurements.

There will be two additional mouse and two additional rat challenge experiments performed and lung

RNA samples prepared.

Milestone 36


Final integration of expression data and informatics analysis

Institution: UNM/

ASU-Johnston


03-01-2009


Pending


Semi-Annual: 10/1/2008 to 3/31/2009



ASU has begun compiling the datasets to identify genes with consistent expression patterns over time (Figure 1, Milestone 35). For integration of data, ASU first compared the microarray expression patterns of genes with defined expression patterns (increasing, decreasing, or stable over the time course after infection) with a list of 28 genes identified as potentially immunoreactive by the ASU TVDC proteome project. To identify the 28 genes by the proteomic approach, approximately 318 protein pools (each pool contained 7 proteins) were prepared by in vitro transcription and translation. The protein pools were tested for their capacity to induce interferon gamma ELISPOTS by lymph node or spleen cells from primates immunized with the

Francisella tularensis LVS vaccine strain. From an initial ELISPOT experiment, 4 of the protein pools were identified as immunostimulatory. This proteomic list of 28 genes (4 pools containing 7 proteins each) were compared to the lists of genes identified via microarray analyses as either upregulated, downregulated, or stable during the 24 hour time course of infection. Genes in common between the proteome and transcriptome approaches were identified (Table 1).

Page 36 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Table 1. Correlation of gene lists with defined microarray expression patterns are cross-referenced with the genes identified by the lymphocyte assays in the ASU TVDC proteomic project. 10 common genes between the proteome and transcriptome approaches are listed in the bottom panel of Table 1.

File Location… R:\GeneVac\FTU\Contract\Microarray\Milestones\36\Gene_Comparison.xls



The next comparison was to create a literature-based list of genes that are associated with virulence or antigenicity. ASU compiled a list of 286 potentially important genes from approximately 10 papers published describing various virulence or immune stimulatory properties of F. tularensis genes. This literature list of genes was cross referenced with the microarray gene lists that were either upregulated over time (Table 2) or downregulated over time (Table 3). These analyses will be used during the final selection criteria for the gene list for protein production for vaccine candidate evaluation.

Page 37 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Table 2. Correlation of the microarray gene list of upregulated genes over the time course of SCHU S4 infection with a literature-based list of genes that have a correlation with virulence of antigenicity. 18 genes correlate between the literature list and the microarray gene list of upregulated genes.

File lo cations … R:\GeneVac\FTU\Contract\Microarray\Milestones\36\Gene_Comparison.xls

Table 3. Correlation of the microarray gene list of downregulated genes over the time course of SCHU

S4 infection with a literature-based list of genes that have a correlation with virulence of antigenicity. 14 genes correlate between the literature list and the microarray gene list of downregulated genes.

Page 38 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



File locations … R:\GeneVac\FTU\Contract\Microarray\Milestones\36\Gene_Comparison.xls


None


Data from two more mouse and two more rat early kinetic of SCHU S4 infection experiments will be generated on MS 35 and analyzed on MS36. ASU requested the RNAs from UNM during the 3/31/09 site visit. UNM will expose the mice and rats to an intranasally delivered dose of approximately 2000

CFU.


None


Good


10%

9. Work plan for upcoming month/ 6 months

For the next month:

ASI will continue with analyses gathered from the two mouse and one rat time course experiments.

For the next 4 months:

The new experiments will be collected and analyzed. As more microarray data are obtained, the results will be integrated into the analyses so that the final results will be the compilation of the total data sets. The goals are to identify by pattern analyses the genes that are reproducibly either increasing in expression, decreasing in expression, or maintaining a steady expression over the first

24 hours of the time course of SCHU S4 infection. In addition, the prior and additional microarray expression data will be integrated bioinformatically with the genes identified in other programs (ASU

TVDC proteomic analyses) and to genes reported in the literature that are associated with virulence or antigenicity.

Milestone 49


Construct single mutants in F. tularensis subsp. tularensis (SCHU S4)

(iglC, pdpD, iglD, iglA, iglB)

49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)

49.2

: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F. tularensis subsp. tularensis (SCHU S4)

49.3

: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F. tularensis subsp. tularensis (SCHU S4)

Institution: UTSA

1. Date started: April 1, 2006




In order to generate mutants in SCHU S4 we need to develop tools to generate successful deletions. Therefore, our focus is twofold, one is cloning experiments to get our target deletions into vectors that we can use in creating these deletions and experiments with SCHU S4 itself using constructs that we believe will allow us to make deletions into SCHU S4.

Page 39 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



I. Cloning:

a. NadM gene mutation using a targetron approach: Cloning of the NadM plasmid was started by having the gene sequence analyzed via Sigma’s algorithm program. The E-value (0.135) indicated position 602/603s was a good insertion site and oligos were ordered. i. The cloning for the intron II NadM construct into pKEK1140 (tulatron) plasmid was completed (pKEK1230). ii. The SchuS4 strain was transformed with this “tulatron NadM” plasmid (pKEK1230) and kanamycin resistant transformants were used to isolate genomic DNA. iii. Polymerase chain reactions using these genomic templates were performed with NadM specific oligos (NadM NcoI and NadM EcoR1) to screen for the correct SchuS4 NadM mutant. Of twenty-two initial screened transformants, we continued with three clones to passage in media or TSA+++ 60 ug/ml kanamycin plates to try and generate a complete

NadM mutant purified away from wild type. Unfortunately, after cycle 6 the complete

NadM mutant has not been generated; the mutant is present in the wild type background.

We continue to cycle these three mutants while working another approach for a new

NadM construct which utilizes a mating plasmid, pJC84 obtained from Dr. Jean Celli’s lab. b. NadM gene mutation using a mating plasmid approach: This new plasmid construct (pJC84) has

Francisella tularensis (Ft) optimized expression markers (i.e. Ft promoter), kanamycin resistance gene, sacB gene for counter-selection on sucrose plates; six unique cloning sites; E. coli origin of replication (so will not replicate in Ft); and is designed for homologous recombination in SchuS4.

We are cloning into this plasmid a transposon T20 (ISFn/FRT- size is 1401 bp) sequence along with the NadM gene sequence. The transposon has a kanamycin cassette flanked with FRT which is the flip recombinase recognition sites and which allow for removal of the kanamycin cassette from the chromosome. In addition, we used a genomic template taken from a F. novicida NadM T20 mutant (tnfn1_pw060328p06q193) obtained from a group at Washington

University. The NadM T20 mutant has the insertion at nucleotide (nt) 565 and nt 566 which is within the C-terminus of the NadM protein. The oligos used (nadM-SalI and nadMSalIStop) amplified the T20 (ISFn/FRT), the NadM sequence and about 600 bp upstream and downstream of this gene (this resulted in 3634 bp sequence). i. As part of the cloning strategy into pJC84 we used an intermediate step where a commercial plasmid pGEM-T was used; this allowed fast cloning of the PCR product mentioned above directly into this vector by using the adenosine overhangs that typically are generated during a PCR. This intermediate step is completed (pKEK1265). We are currently working on cloning the entire 3634 bp T20 NadM fragment into pJC84. c. FTT0748 gene mutation using a targetron approach: We started the cloning of the final gene target FTT0748 where we sent sequence for analysis to Sigma and selected 561/562s as the optimal location to insert the intron II FTT0748. The oligos were order and used to amplify the intron II FTT0748 and after digestion of both pKEK1140 and this product with BsrGI/XhoI we proceeded with a ligation and subsequent transformation of DH5α cells. This month’s report will discuss results of this transformation.

II. Schu S4 Experiments: a. The VgrG SchuS4 mutant (KKF13) had been tested for its virulence in September and we checked for this mutant’s ability to protect the mice against challenge with wild-type SchuS4

(KKT1). The various vaccinated mice groups (5 each) were inoculated by intranasal route with a dose of 81 CFUs. The various VgrG SchuS4 vaccinated groups (1.988 E3 up to 1.988 E6) died by day 5, as did the naive mice. Therefore, no protection was seen with this attenuated mutant.

III. We are continuing to passage potential NadM Schu S4 mutants generated from a transformation made with the “tulatron construct”, pKEK1230. We are currently on passage 6 and 7 and have not found a complete Schu S4 mutant by our PCR screening using oligos that anneal to the 5’ and 3’ end of the NadM gene (nadM-NcoI and nadM-

EcoR1). This oligo set will generate a ≈1900 bp size product for the mutant strain; the

Page 40 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel wild type PCRs product would be ≈1000 bp. We continue to see a mixed population from isolated genomics of various passaged isolate strains.


I. Cloning:

a. Continued with the cloning of the intron II FTT0478 gene sequence into pKEK1140. The DH5α cells had been transformed in last report so we prepared plasmid isolations from 10 clones which resulted from this transformation. Subsequently, these plasmid preparations were digested with

Bgl II restriction endonuclease and the results are in figure 1.

Figure 1.

1 Kb

4.0

1.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Legend:

1. 1 Kb Ladder 8. pKEK1140

2. C1 1140+738 9. C6 1140+748

3. C2 1140+738 10. C7 1140+738

4. C3 1140+738 11. C8 1140+738

5. C4 1140+738 12. C9 1140+738

6. C5 1140+738 13. C10 1140+738

7. Uncut pKEK1140 14. 1 Kb Ladder

Figure 1 represents a Bgl II digestion profile of various intron II FTT0748 plasmid constructs (lanes 2-6, and 9-13) from a transfo rmation of DH5α cells and control pKEK1140 plasmid (lane 8). The correct plasmid will illustrate a downward shift of the largest DNA band compared to the control, pKEK1140 digested profile (≈3850 bp). This shift is approximately a 450 bp decrease (to ≈3400 bp) and is not so obvious in this gel however, C6 1140+748 (lane 9) is directly next to the control plasmid and there is an evident decrease in the largest band. Although all these generated transformants may be correct, we decided to proceed only with C6 1140+748 plasmid. Data located in TVD UTSA Notebook 7, page 83. b. The transformant C6 1140+748 which contains the potentially correct intron II FTT0748 plasmid was used to start a 100 ml LB+50 ug/ml kanamycin culture to grow overnight.

These cells were harvested and used to prepare a midi plasmid isolation using the

Qiagen midi plasmid kit. This plasmid was mailed off for sequencing to Lone Star Labs in

Houston. Results of sequencing will be noted on next report. c. Continued with the cloning of the T20 NadM transposon into pJC84 using the pKEK1265 plasmid generated earlier. Our first attempt to clone this T20 NadM transposon sequence (“insert”) from pKEK1265 into pJC84 was by using Sal I as the restriction site to access the pJC84 plasmid. This resulted in all re-ligation of the pJC84 plasmids without the insert. Therefore, as discussed in last month’s report, we used the cloning sites Sac I and Sma I in the pJC84 plasmid and the Sac I and Nco I in the pKEK1265 plasmid. The Sma I creates a blunt end and by treating the Nco I digested fragment with

Klenow enzyme treatment we can create a blunt end which can then be used to ligate to the Sma I portion of the pJC84 plasmid. These restriction endonucleases were used on the appropriate plasmids in digestion incubation at 37ºC overnight. Once each of these plasmids were manipulated with the appropriate enzymes we ran these digested plasmids on a 1% agarose gel (in 1X TAE buffer) and band isolated the respective DNA components needed for the ligation of pJC84 plasmid (≈3600 bp) with the T20 NadM sequence from pKEK1265 (≈3800 bp). This ligation was used in a transformation experiment with DH5α cells which were spread onto LB 50 ug/ml kanamycin agar plates as the final step. After incubation at 37C overnight this transformation yielded hundreds of colonies.

Page 41 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel d. The pJC84 plasmid and the intended DNA component (T20 NadM sequence) both have a kanamycin cassette in their respective DNA so we have no way to counter-select for

DH5α transformants containing only pJC84 + T20 NadM sequence compared to pJC84 re-ligations. However, typically when directional cloning is done the ratio of re-ligation events versus the cloned insert into the pJC84 will have a much higher frequency by 30 to 60% more transformants. The control pJC84 re-ligation did in fact have at least 30% less transformants so we should be able to obtain our correct construct from this set of transformed cells. e. We randomly selected 10 colonies from the pJC84+T20 NadM sequence ligation transformants and used them to prepare mini plasmid isolations. These plasmids were digested with two different enzymes, respectively, to determine if the T20 NadM sequence is present in any of these selected colonies (figure2).

Figure 2.

1 Kb

4.0

2.2

Pst I

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Legend:

1. 1 Kb ladder

2. Uncut pJC84

8. C5 pJC84+T20 NadM

9. C6 pJC84+T20 NadM

3. pJC84 10. C7 pJC84+T20 NadM

4. C1 pJC84+T20 NadM 11. C8 pJC84+T20 NadM



7. C4 pJC84+T20 NadM 14. Uncut C5 pJC84+T20 NadM

3.2

2.0

BamHI

Figure 2 represents two individual restriction endonuclease digestions of various pJC84+T20 NadM plasmids (lanes 4-13). The loading order of these plasmid for the top and bottom of this gel is identical however, the top panel represents Pst I digested profiles and the bottom panel represents BamHI digested profiles. The BamHI profile (lower panel) should yield a linearized profile for both the control pJC84 plasmid and the correct construct. Therefore, the correct plasmid should be at ≈7400 bp instead of ≈3600 bp

(pJC84) when compared to control. The BamHI enzyme did not cut the various plasmids to completion as seen by comparing the uncut plasmid profiles (lanes 2 and14) to the digested profiles. However, it is clear that none of these select ed colonies had the correct plasmid since there is no strong DNA bands at ≈7400 bp. The Pst I digestion profile should have shown the pJC84 plasmid control linearized (≈3600 bp) but the correct T20 NadM plasmid should have yielded two DNA bands. The size of the two bands for the correct plasmid with the Pst I digestion depends on the orientation of the original T20 NadM plasmid (pKEK1265).

We should see either a 6859 bp with 541 bp or a 5708 bp with a 1692 bp DNA band. Based on the presence of a single band retained in the Pst 1 digestions, it is clear that this digested verified only religation in this selected group. Data located in TVD UTSA Notebook 7, page 87. To date, the NadM gene plasmid has not been generated using the mating plasmid approach. Therefore, this approach will be continued only up to the point that we get the NadM mutant in Schu S4 or we have the mating plasmid completed and ready to use with Schu S4; whichever comes first.

II. Schu S4 Experiments: a. In the last experiments, we screened thirty cycle 5 and cycle 6 NadM Schu S4 isolates by

PCR using oligos that anneal to the 5’ and 3’ end of the NadM gene (nadM-NcoI and nadMEcoR1 {“forward and reverse nadM oligos”}). We only found mixed populations from these prepared genomic isolates; however, a few PCR profiles did indicate that the mutant size band (≈1900 bp) was becoming more evident compared to its parent /wild type genomic isolate PCR profile. So only a few of these 30 strains were used to continue with passaging on TSA+++60 ug/ml kanamycin plates. These clones were identified as N2F,3-1E, N3-4B,4-33g, N3-4,4-33h, N7-6,4-23A and N7-6,5-3D.

Page 42 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel b. Each of the selected 9 clones where streaked for single colonies on the above mentioned plates and the isolated colonies that resulted were patched onto fresh TSA+++60ug/ml kanamycin plates. These patched clones were used to isolate their genomic DNA and

Figure 3. again the PCR experiment was repeated using the forward and reverse NadM oligos

(figure 3).

1 Kb

2.0

0.8

1 2 3 4 5 6 7 8 9 10 11 12 13

Legend:

1. 1 Kb Ladder 15. N3-4B,4-33g2

2. KKT1 16. N3-4B,4-33g3

3. N2F,3-1E1

4. N2F,3-1E2

5. N2F,3-1E3

6. N2F,3-1E4

7. N2F,3-1E5

8. N7-6,5-3D1

9. N7-6,5-3D2

10. N7-6,5-3D3

11. N7-6,5-3D4

12. N7-6,5-3D5

13. N7 orig

14. N3-4B,4-33g1

17. N3-4B,4-33g4

18. N3-4B,4-33g5

19. N3-4B,4-33g6

20. N3-4B,4-33H1

21. N3-4B,4-33H2

22. N3-4B,4-33H3

23. N3-4B,4-33H4

24. N3-4B,4-33H5

25. N3-4B,4-33

2.0

0.8

1 2 14 15 16 17 18 19 20 21 22 23 24 25

Figure 3 represents the PCR profiles of various cycled NadM Schu S4 mutant candidates using the forward and reverse NadM oligos. The correct Schu S4 NadM mutant should yield only one PCR product of ≈1900 bp. The wild type profile (lane 2) will show ≈1000 bp PCR product. Lanes 3 thru 12 and 14 thru 24 represent isolated Schu S4 NadM clones that were on cycle 6 or 7. Lane 13 is N7 original clone and lane 25 a previously cycled isolate N3-4B,4-33 (this would have been passage 5 clone). This gel profile is not very clear; however, it is evident that there are no complete NadM Schu S4 mutants without wild type profile present. The comparison with the parent (lane 13) or a previously cycled clone (lane 25) with this new test group show that a few may be showing a stronger mutant product and we will continue with passage with some of these (perhaps, N2F,3-1E3, N3-4B, 4-33g5, N3-4B, 4-33h3, and N7-6,5-3D4 from this test group).

Data located in TVD UTSA Notebook 7, page 81.


NadM is a metabolic protein and we have been able to create a mutant in F. novicida NadM mutant using the “tulatron plasmid construct”. In addition, an independent group also created a NadM mutant using a different type of transposon T20 (ISFn/FRT) in F. novicida so we are hopeful that our lab will be able to create this mutant in SchuS4 by the same means. It is possible that NadM gene is critical in

SCHU S4 and a NadM SCHU S4 mutant cannot survive. If these transposon insertions in the NadM gene remain problematic in SchuS4 we may choose a different gene target named atpC. This gene is a regulator in a metabolic pathway in SchuS4.


KKF5: igLC1 IgLC2 Schuh4: KKF10: iglD1 igLD2 Schuh4; and KKF13: VgrG1 VgrG2 Schuh4 mutants are completed Schuh4 strains to date.


Good


92%

9. Work plan for upcoming month and next 6 months

Next month

Page 43 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel a.

Will continue with the selection of NadM mutant in SchuS4 using the transformants’ passaged clones f rom the pKEK1230 plasmid (“tulatron”) b. Will continue the cloning of the new transposon NadM into pJC84 plasmid using the T20 NadM sequence from the pKEK1265 (pGEM-T+T20 NadM).

Next 6 months: d. Will finish the NadM Schu S4 mutant and verify by PCR and subsequent sequencing. e. Will characterize the NadM Schu S4 mutant by checking intra-macrophage survival and potential attenuation in mice. f. Will use the intron II FTT0748 tulatron plasmid to transform Schu S4 and create the

FTT0748 mutant. Then verify by PCR and subsequent sequencing. g. Will verify the NadM, and FTT048 further by a Southern blot using igLC and igLD SchuS4 mutants as added controls for this experiment. h. Will further characterize the FTT0748 mutant by checking intra-macrophage survival and potential attenuation in mice. i. Both the NadM and FTT0748 Schu S4 mutants, respectively, will be also checked for their ability to protect mice after vaccination with a challenge of these vaccinated mice with wild type Schu S4 strain. j. If problems are encountered with the NadM SchuS4 mutant will proceed with the atpC metabolic gene as the alternate target mutation in this strain.

Milestone 50


Phenotyping and confirmation of single gene mutants;

50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB ; LVS uvrA or uvrB , and Ft subsp. tularensis (SCHU S4) iglC strains,

50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD, iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,

50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA, iglB strains

Institution: UTSA


04/01/2006


provide date when milestone is completed


Semi-Annual: 10/1/08 to 3/30/09-SUMMARY/HIGHLIGHTS

(50A) We evaluated the protective efficacy of two Ft subsp. tularensis mutants (

 iglD and

 vgrG) and the Ft novicida iglB mutant against pulmonary Type A SCHU S4 challenge using a murine tularemia model. a. SCHU S4



iglD (KKT10) i. Mice orally vaccinated with KKT10 (10 3 CFU) produced significant amounts of systemic (serum) and mucosal (intestinal) antigen-specific antibody. ii. Mice vaccinated orally with this mutant did not protect from subsequent lethal pulmonary challenge with wild type SCHU S4 (20-400 CFU), although a prolonged median survival time was observed. b. SCHU S4



vgrG (KKT13) i. This mutant is highly attenuated as it did not replicate in J774 macrophages and had a LD

50

greater than 10 6 CFU when challenged intranasally. ii. Mice orally vaccinated with KKT13 (10 3 CFU) produced significant amounts of systemic (serum) and mucosal (intestinal) antigen-specific antibody.

Page 44 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel iii Mice vaccinated orally with this mutant did not protect from subsequent lethal c. U112 pulmonary challenge with wild type SCHU S4 (30-150 CFU)



iglB (KKF235) i. Mice orally vaccinated with KKT13 (10 3 CFU) produced significant amounts of systemic (serum) and respiratory antigen-specific antibody.

(50B) We completed the last few experiments to close out this sub-milestone from 10/1/08-

11/30/08. a. Cellular responses to oral LVS vaccination 8 and 12 weeks post-challenge i. Splenocytes isolated from mice 8 and 12 weeks after oral vaccination with LVS still secreted IFN-



upon secondary stimulation with higher doses of UVinactivated LVS. However, this response was diminished compared to spleens isolated 2 and 4 weeks after vaccination b. Role of CD8+ T cells in protection conferred by Oral LVS vaccination i. Depletion of CD8 + T cells at time of challenge abrogated protection conferred by oral LVS vaccination and resulted in 100% mortality of mice. This suggests that these cells play an important role in clearance of the infection. c. Long term protective efficacy of oral LVS vaccination

i. Mice which received a secondary booster dose of LVS orally exhibited enhanced protection against a high SCHU S4 challenge dose compared to mice which received only one vaccination dose.


50A: Evaluate the protective efficacy of oral KKF235 ( ΔiglB of U112) vaccination against

SCHU S4 intranasal challenge. (Note book #9, page 27-28). Mice were given orally a single dose (10 3 CFU) of KKF235 or LVS, rested for 30 days, and challenged intranasally with 34

CFU of Type B (OR96-0246 strain). Mice receiving PBS were used as a mock control and all succumbed to lethal SCHU S4 challenge by day 10. All LVS vaccinated mice survived the lethal SCHU S4 challenge dose by day 30. Mice vaccinated with KKF235 ( ΔiglB of U112) had a 17% survival rate and a prolonged media survival time of 9.5 days when compared to 6 days for the PBS mock vaccinated mice ( p <0.005 in a log rank test using Kaplan-Meier survival analysis). In summary, oral immunization of mice with F. novicida iglB mutant, although not as potent a vaccine as LVS, did provide significant protection against lethal

Type B F. tularensis pulmonary challenge when compared to PBS mock immunization.

Page 45 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



100

80

60

40

20

0

100

0

80

60

40

20

0

0

5 1 0 1 5

LV S

PB S (mock)

2 0 2 5

K K F235

P B S (mock)

3 0

5 10 15 20

Days post challenge

25 30

Fig. 1. Protective efficacy of KKF235 ( iglB mutant of U112) immunization against Type B F. tularensis infection. Mice were mock vaccinated with PBS or orally immunized with 10 3 CFU of KKF235 or LVS and i.n.

challenged with lethal dose (34 CFU) of OR96-0246 strain .

Mice were monitored for survival.

(Note book #9, page 27-28).


None


None


None


Good


100% of scientific work completed on milestone 50A (original plans)


None, this milestone is closed on 03/31/2009.

UTSA will write the MSCR for MS 50A

Milestone 52


Create RecA mutants in F. tularensis subsp. tularensis(Schu S4)

Institution: UTSA





a.

NR5330 (FTN0720) and NR7241 (FTN0757) are the F.novicida transposon mutants. The gene FTN0720 functions as transcriptional regulator, and FTN0757 is known as the membrane protein of unknown function. Our goal in this study was to evaluate the attenuation

Page 46 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel of these two F. novicida mutants in Balb/C mice and subsequently protective efficacy of the mutants against wild type F.novicida challenge. i. Confirmation of the transposon insertion. The two mutants were confirmed by screening the transposon insertion in the target gene using PCR with the transposon specific primer, and the PCR product was sequenced. Both PCR and sequencing data verified the transposon insertion in the target gene for both NR5330 (FTN0720) and NR7241

(FTN0757). ii. Evaluation of attenuation in the Balb/C mice. The mice survived after 30 days post vaccination intranasally with the dose 200CFU for NR5330 and 534CFU for NR7241 except for one (NR5330) which died 11 days after inoculation. Both of the transposon mutants were attenuated in Balb/C mice. iii. Evaluation of protective efficacy of the mutants against wild type Ftn U112 challenge in the survival mice. The mice survived after 30 days post challenge with the dose of 242

CFU by intranasal inoculation. Both mutants provided protection in Balb/C mice against wild type F. novicida U112 challenge.

b.

Creation of recA and IglC double mutants in F. tularensis tularensis (SCHU S4). Inactivating the recA gene can stabilize the strain and prevent the strain from any additional genetic changes. KKT5 was the IglC mutant Schu S4 and parent strain in this study. The tulatron vector pKEK1186 was used as the tool to disturb and inactivate the recA gene in KKT5. i. Delivery of pKEK1186 to KKT5. The tulatron vector pKEK1186 was transformed into the parent strain KKT5 by cryotransformation. The potential transformants were selected on

TSA++ agar plate with 50ug/ml Kanamycin at 30°C since pKEK1186 carried Kanamycin marker. ii. Determination of the mutated intron insertion in the potential transformants. Two sets of colony PCR were performed using recA gene specific primers or the combination of recA gene specific primer and insertion specific primer to confirm the intron insertion in recA gene of KKT5. Both PCR reactions confirmed the insertion in the screened transformants. iii. Separation of recA mutant KKT5 from KKT5. The transformants confirmed by PCR were streaked onto TSA++/Kanamycin agar medium and incuba ted at 30°C to obtain the single colonies which were screened using PCR mentioned in Step ii. This procedure was repeated until the pure mutants were obtained and no wild type KKT5 remained.

iv.

Removal of the plasmid from recA mutant KKT5 at 37°C. The tulatron vector was the temperature sensitive plasmid, which can be removed from the bacteria as the temperature is changed from 30°C to 37°C. The recA mutant KKT5 without the plasmid was screened using the same PCR reaction as mentioned above, and confirmed by sequencing of PCR product. The recA mutant KKT5 was designated as KKT23.

c.

Creation of restriction enzyme (FTT1579 and FTT0523) mutants in Schu S4. This goal is to break down the restriction barriers (FTT1579 and FTT0523) of Schu S4, which limit the entrance of foreign plasmid DNA into Schu S4 strain. The method allows us to retarget these two restriction sites and inactivate certain gene(s) to facilitate the introduction of foreign plasmid DNA into Schu S4 strain. i. Construction of tulatron vector for inactivating FTT1579 (same as FTN1487) gene in

Schu S4 (Ftn U112). The insertion sites were selected at 849/850bp or 1254/1255bp for

FTT1579 (FTN1487), and 390/391bp for FTT0523 gene disruption. The primers for

FTT1579 (FTN1487) at 849/850bp or 1254/1255bp insertion sites were ordered and PCR amplification for producing 350bp PCR fragment to mutate intron RNA was performed.

Monthly report—March, 2009

3.1 Creation of RecA and IglC double mutant in F. tularensis tularensis (SCHU S4).

Page 47 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



This part of Milestone 52 is to create recA and IglC double mutant in F. tularensis tularensis. Inactivating the recA gene will stabilize the strain and prevent the strain from any additional genetic changes. We already have the IglC mutant of Schu S4, and the tulatron vector pKEK1186 for disturbing and inactivating the recA gene in Francisella tularensis .

3.1.1 In last monthly report, we reported that 5 colonies were obtained from which the plasmid had been removed, but we needed to screen these colonies using PCR to assure the insertion in recA gene. The colony PCR was set up as follows:

5XGreen GoTaq Buffer 4.0ul dNTPs mix, 10mM each 0.4ul recA Schu4 for (25pmol/ul) 1.0ul

EBS Universal or recA Schu4 rev (25pmol/ul) 1.0ul

GoTaq DNA polymerase 0.1ul

DNA 1.0ul

DNAse,RNAse free water 12.5ul

At 95°C 2 min, 95°C 30 sec/55°C 30 sec/72°C 1min40sec//30 cycles, 72°C 5 min

Figure1: Gel picture of colony PCR using recA gene primers “recA Schu4 for” and recA

Schu4 rev”.

Figure1 legend, results and data location: Lane5 (about 630bp) was the parent strain KKT5 as the negative control, and lane6 (about 1.5kb) was KKF348 (recA mutant U112) as the positive control. With the intron insertion in recA gene of the mutants, PCR product was about 900bp larger than the parent strain KKT5. All of 5 colonies (lane2-lane4 and lane7-lane8) had the same size band as the positive control, which confirmed that the intron insertion existed in recA gene of the SCHU S4 strain.

Data recorded on UTSA TVDC notebook #6, page64 for Figure1.

Page 48 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Figure2: Gel picture of colony PCR using the insertion specific primer “EBS Universal” and gene primer

“recA Schu4 for”.

Figure2 legend, results and data location: Lane5 was the negative control (KKT5) without any band presenting, and lane6 was the positive control (KKF348) with the band at about 0.5kb. Lane2-4 and lane7-8 were 5 colonies screened which had the same results as the positive control.

Data recorded on UTSA TVDC notebook #6, page65 for Figure2.

The data presented in figure1 and 2 supported that colony8-1, 8-2, 8-3, 8-26, and 8-29 were the recA mutant KKT5 (recA and IglC double mutant Schu S4) with the plasmid removed.

3.1.2 The purified PCR DNA from lane7 (colony8-26) on figure2 was sent for sequencing with the primers “EBS Universal “ and “recA Schu4 for”, and the sequencing data further confirmed the intron insertion in recA gene of this strain. Colony8-26 was designated as KKT23, and the frozen stocks were made for permanent storage.

Data recorded on UTSA TVDC notebook #6, page67-68 for the sequencing result.

3.2 Creation of FTT1579 and FTT0523 gene mutants in Schu S4. FTT1579 functions as

Type III restriction enzyme, and FTT0523 as the hypothetical protein that is similar to

Q89Z57 Type I restriction enzyme EcoAI specificity protein. Both of FTT1579 and

FTT0523 genes limit the entrance of the plasmid DNA into Schu S4 strain. This goal is to break down the restriction barriers (FTT1579 and FTT0523) of Schu S4. The method allows us to retarget these two restriction enzymes and inactivate certain gene(s) to facilitate introduction of the plasmid DNA into Schu S4 strain.

3.2.1 We reported that 849/850bp was selected as the insertion site for FTT1579 or

FTN1478 gene disruption in last monthly report. In case 849/850bp insertion site does not work well, another insertion site 1254/1255bp was selected for the alternative of 849/850bp insertion site. The primers for 849/850bp insertion site were listed below:

849/850aIBS: 5’-AAA ACT CGA GAT AAT TAT CCT TAT GAG TCT ATT TGG

TGC GCC CAG ATA GGG TG3’

948/850aEBS1d: 5’-CAG ATT GTA CAA ATG TGG TGA TAA CAG ATA AGT CTA

TTT GCT TAA CTT ACC TTT CTT TGT3’

849/850aEBS2: 5’-TGA ACG CAA GTT TCT AAT TTC GGT TAC TCA TCG ATA

GAG GAA AGT GTC T3’

The primers for 1254/1255bp insertion site were shown as follows:

Page 49 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



1254/1255sIBS: 5’-AAA ACT CGA GAT AAT TAT CCT TAT TTA ACG ATG GCG

TGC GCC CAG ATA GGG TG3’

1254/1255sEBS1d: 5’-CAG ATT GTA CAA ATG TGG TGA TAA CAG ATA AGT

CGA TGG CAA TAA CTT ACC TTT CTT TGT

1254/1255sEBS2: 5’-TGA ACG CAA GTT TCT AAT TTC GGT TTT AAA TCG

ATA GAG GAA AGT GTC T3’

The universal primer for each insertion site was:

EBS Universal: 5’- CGA AAT TAG AAA CTT GCG TTC AGT AAA C-3’

3.2.2 Made 4-primer master mix for PCR with IBS, EBS1d, EBS2, and EBS Universal primers. Performed PCR to get 350bp product with 4-primer mix for both

849/850bp and 1254/1255bp insertion sites. PCR was Set up as follows:

23ul ddH2O

1.0ul 4-primer mix

1.0ul Intron PCR template

25.0ul JumpStart RedTaq Ready mix

At 94



C 30sec, 94



C 15sec/55



C 30sec/72



C 30sec//30 cycles, then 72



C 2min.

Figure3: Gel picture of 350bp PCR.

Figure3 legend, results and data location: Lane2 was PCR for 849/850bp insertion site and lane3 was for

1254/1255bp insertion site. There were 2 bands in each lane. The smaller band was 350bp PCR product for retargeting intron RNA.

Data recorded on UTSA TVDC notebook #6, page 72 for figure3.


None

5.

Problems or concerns and strategies to address

None


KKT23 (RecA and IglC double mutant Schu S4)


Good


.

About 55% of scientific work completed.

9. Work plan for upcoming month and 6 months

For next month: a. Purify the parent plasmid pKEK1140 and 350bp PCR products . b. Digest pKEK1140 and 350bp PCR product with BsrGI and XhoI. c. Ligate 350bp PCR into pKEK1140.

Page 50 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel d. Transform ligation reaction into host cells.

For next six months: a. Construction of the tulatron vector for FTT1579 (FTN1487) gene mutagenesis. b. Creation of FTN1487 (FTT1579) gene mutants in Ftn U112 (Ftt Schu S4). c. Construction of the tulatron vector for FTT0523 gene inactivation. d. Creation of FTT0523 gene mutant in both wild type Schu S4 and FTT1579 mutant Schu S4.

Milestone 53B


Examining the protective efficacy of LVS and two attenuated SCHU S4 mutant strains via oral vs. intradermal inoculations in the rat model;

50.1: replication of LVS, Schuh4, iglC Schuh4, and one additional attenuated Schuh4 mutant derived in milestone 49 in rat macrophages .

50.2: protective efficacy of LVS, iglC Schuh4, and one additional attenuated Schuh4 mutant derived in milestone 49 against Schuh4 intratracheal challenge (oral vs. intradermal vaccinations in rats)

50.3: antigen specific cellular and humoral responses of rats following vaccination with LVS, iglC Schuh4, and one additional attenuated Schuh4 mutant derived in milestone 49

50.4: bacterial dissemination and lung pathology of rats following vaccination with LVS, iglC

Schuh4, and one additional attenuated Schuh4 mutant derived in milestone 49

Institution:

UTSA


2. Date completed: provide date when milestone is completed


Semi-Annual: 12/1/08 to 3/30/09-SUMMARY/HIGHLIGHTS

(53B) Macrophages were derived from the bone marrow of F344 rats and evaluated for purity and basal phagocytic activity. We then evaluated the capability of several Francisella strains to replicate within this rat bone marrow cell type. a. Viability of F344 bone marrow derived macrophages i. Through flow cytometric analysis, macrophages were cultured from rat bone marrow at a purity of 95%. ii. Using fluorescent micro beads, we found that the basal phagocytic activity of these rat bone marrow derived macrophages was greater than that of bone marrow macrophages similarly derived from BALB/c mice. b. F. novicida phagocytosis i. This strain was readily picked up in large quantities by Fisher 344 rat bone marrow derived (BMD) macrophages. Bacteria were able to replicate from 1-2 logs in these cells within the first 24 hours after which infection was controlled. c. LVS phagocytosis

i. This strain was only present intracellularly in very small numbers immediately following infection of the rat BMD macrophages. Subsequently, LVS bacteria were unable to replicate over time. d. F. holarctica phagocytosis

i. This strain was also present in very low numbers following infection of the rat BMD macrophages. However, unlike LVS, these F. holartica bacteria were able to slowly replicate throughout the entire time course which resulted in a gain of 3-4 logs.

Page 51 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009




53B-a: (1) Replication of F. novicida U112 and F. tularensis SCHU S4 within rat bone marrow derived macrophages. (Note book # 10, pages 30, 33, 37, 41). Bone marrow derived macrophages were derived from Fisher 344 rats, seeded in 96-well culture plates at a density of 2

X 10 5 cells per well and allowed to adhere over night. Cells were infected with either F. novicida or F. tularensis SCHU S4 at 10 and 100 MOI for 2 hours. Cells were then pulsed with Gentamicin for 1 hour to kill any remaining extracellular bacteria, after which they were incubated at 37 degrees C. Cells were lysed at 3, 24, 48, or 72 hours after infection and serial dilutions of lysate were plated on TSA plates to enumerate intracellular bacteria. As shown in Figure 1, there was an initial uptake of 10 3 -10 4 CFU of F. novicida at 3 hours followed by 1-2 logs of replication by 24 hours post-infection. From 48 to 72 hours after infection, the numbers of viable F. novicida slowly decreased. In contrast, only 10 2 – 10 3 CFU of F. tularensis SCHU S4 were taken up by the macrophages initially. Similar to findings previously reported with LVS (see report Jan 09), there was minimal replication seen with SCHU S4. This experiment has been repeated twice with similar results. The inability of SCHU S4 to replicate inside rat macrophages needs to be further clarified.

F. nov ic ida

10 7

10 6

10 5

10 MOI

10 7

10 6

10 5

100 MOI

10 4 10 4

10 3 10 3

10 2 10 2

10 1

10 1

3 24 48 72 3 24 48 72

H ours After Inoc ulation

Fig. 1. Intramacrophage growth of F. novicida and SCHU S4 in rat BMDM. Primary bone marrow derived macrophages derived from Fisher 344 rats were infected with F. novicida U112 or F. tularensis SCHU S4 at either 10 or 100 MOI. Cells were lysed and viable bacteria were counted at 3, 24, 48 and 72 hours after infection.


None


None


None


Good


15%

Page 52 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009




Next month

53B.

(1) Assess the need for opsonization of SCHU S4 and/or LVS to replicate within F344 bone marrow derived macrophages.

(2) Serum antibody titers of F344 rats following either oral or intradermal LVS vaccination

Next 6 months

53B. (1) Investigate the requirement of opsonization for increased phagocytosis and replication of

LVS and SCHU S4 strains

(2) Evaluate humoral and cellular responses after both oral and intradermal vaccination with

LVS

(3) Evaluate the protective efficacy of oral and intradermal vaccination with LVS followed by

SCHU S4 challenge

(4) Bacterial dissemination following oral and intradermal vaccination with LVS

Page 53 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



Sections IV: Contract Expenditures and Subcontractor Billing

1. % work completed vs. %cumulative costs incurred to date with regard to planned effort and proposed budget

The active Milestones over the past 6 months have been: 2,3, 4, 5, 7, 8, 9, 10, 11(UNM &LBERI), 12/13

(UNM &LBERI),14, 17, 18, 19, 21(UNM &LBERI), 26, 27, 28, 29 (UNM &LBERI), 35 (UNM/ASU), 49,

50, 51, 52, 53, 55, 56, 57.

For the past 6 months, NIAID has been billed for scientific activity on the above listed Milestones.

Data associated with most current UNM invoice submitted to NIAID ( Subcontractors’ data parallels

Financial Report by Milestone associated with UNM Invoice 40A to NIAID and lags behind April 2009 Technical

Report)

Milestone % invoiced % work completed

Over budget?

Discrepancy?

17-UNM

18-UNM

19-UNM

21-UNM

21-LBERI

25-ASU

26-ASU

27-UNM

28-ASU

29-LBERI

29-UNM

32-ASU

33-ASU

2-UNM

2-LBERI

3-LBERI

4-LBERI

5-UNM

7-LBERI

8-LBERI

9-LBERI

10-LBERI

11-LBERI

0

13.6

11-UNM 38.3

12/13 UNM 85.7

12/13 LBERI 65.2

14-LBERI 0

14-UNM

16-UTSA

11.3

100

0

87.4

100.8

82

94

97.8

8.4

2.4

0

0.2

80

93

3.2

100

100

100

103.9

0.7

7.1

100

100

0

5

25

59

2

100

100

100

100

2

15

100

100

0

6

40

72

88

0

5

100

75

73

100

98

99

90

25

40

No

No

No

No

No

No

No

No

Yes

No

No

No

No

No

No

No

No

No

No

No

No

No

No

Yes

No

No

No

No

No

See explanation below #1

NA

Minimal overspend, MS completed

NA

NA

NA

Explanation needed; see #2 below


NA

NA

NA

NA


NA

NA

NA


NA



NA

NA

NA


Minimal overspend; MS completed

NA

NA

NA

NA

Page 54 of 57


Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



34-UNM

34-ASU

35-ASU

35-UNM

39-UTSA

40-Cerus

41-Cerus

42-Cerus

43-UTSA

44-Cerus

46- Cerus

48-UTSA

49-UTSA

50-UTSA

51-UTSA

52-UTSA

100

100

100

100

90.4

91.8

94.8

35.5

100

100

73.8

18.3

100

100

100

100

100

5

53

100

84

90

100

46

100

100

74

25

100

100

85

25

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No


NA

NA

NA

NA

NA

NA


NA



NA

NA

NA

NA

NA

53-UTSA

55-Cerus

56-Cerus

57-Cerus

2.6

37.6

29.3

11.1

4

50

30

12

No

No

No

No

NA

NA

NA

NA

Discrepancy Explanations

1. UNM Discrepancies

MS 2- UNM MS 2 (LVS vaccinations for relevant personnel) has no budget. The expenses for MS 2 consist of the UNM administrative oversight of the LVS vaccinations and also the travel and health screenings for the LVS vaccinees. The administrative costs are not covered under Milestone budgets.

The travel and health screening costs are included under the “Occupational Health” expense category, which was not included in the Milestone budgets. To date, 37 UNM and LBERI staff received the LVS vaccination at USAMRIID.

MS 17-UNM: Previously UNM reported 83.3% invoiced ($87,306) and 25% work completed on the

3/15/09 report submitted to NIAID. Subsequently, Drs. Lyons and Wu realized that the rat model work and expenses should have been charged to MS11 and not to MS 17. UNM moved the $87,306 of expenses from MS 17 and to MS 11 as of the 4/15/09 semi-annual report. As of 4/15/09, UNM reports

0% invoiced and 0% work completed on MS17.

MS 19-UNM: UNM is developing assays with human alveolar macrophages and the limited number of cells collected is challenging. UNM is likely to end this milestone by 7/1/2009. Previously UNM reported

102.8% invoiced ($82,484 spent) and 20% work completed on the 3/15/09 report submitted to NIAID.

UNM moved $19,430 of unspent funds from completed MS27 and $14,254 of unspent funds from completed MS34 into MS 19’s budget; the total moved into MS19 is $33,684. This increase in the MS19 budget, dropped the % invoiced to 80% and the % work completed remains 25%.

MS 21- UNM: UNM is developing macrophage killing assays in vitro in humans and rats. The discrepancy between the 93% invoiced and 59% work completed is due to the developmental aspects of the assays. UNM anticipates that the % work completed will catch up as the % invoiced completed moves to 100%.

MS 27- UNM: MS27 was completed when the peptide /cellular immunology assays were developed with

ASU. UNM moved $19,430 of funds remaining under MS27 and moved the funds into MS19 which was

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Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009


Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel running over budget as of 4/13/09. Moving the unspent funds from MS27, resulted in 100% invoiced as of the 4/15/09 semi-annual report.

MS34- UNM: UNM’s efforts on MS34 were completed when ASU had sufficient RNAs for pilot studies to develop the microarrays for assessing FT in vivo infections in animal host lungs. UNM moved $14,254 of funds remaining under MS34 and moved the funds into MS19 which was running over budget as of

4/13/09. Moving the unspent funds from MS34, resulted in 100% invoiced as of the 4/15/09 semi-annual report.

2. LBERI Discrepancies

MS8-LBERI: LBERI is performing studies to validate LVS vaccination protection of aerosol SCHU S4 in primates. LBERI anticipates that as the vaccination studies proceed the % invoiced will catch up as the

% work completed moves to 100%.

MS9-LBERI: LBERI is qualifying the bioaerosol system for the FT exposures in order to generate an aerosol SOP for GLP transition. This milestone is ahead of schedule since most of the work performed to date has been paperwork that has lower costs. Lab work will begin shortly which will bring the % invoiced in line with the % work completed.

MS12/13-LBERI: LBERI is developing assays for detecting relevant immune responses in animals & humans to compare the responses to those in other species. This milestone is almost complete unless additional assays are identified. The number of assays that would need to be conducted in this milestone were estimates and those identified are almost done. Therefore LBERI expects that the budget will not be 100% expended.

3.CERUS Discrepancies

MS 42- Cerus: 100% Invoiced and 25% work. Cerus terminated MS42 and shifted residual funds into new milestones 55 to 59. UNM and NIAID approved this action in a subcontract modification with Cerus

MS44-Cerus: 100% Invoiced and 5% work. Cerus terminated MS44 and shifted residual funds into new milestones 55 to 59. UNM and NIAID approved this action in a subcontract modification with Cerus

MS46-Cerus: 100% Invoiced and 53% work. Cerus terminated MS42 and shifted residual funds into new milestones 55 to 59. UNM and NIAID approved this action in a subcontract modification with Cerus

To date, administrative costs have been billed to NIAID that are associated with Milestone 2 and with the management of the scientific milestones

2, 3, 4, 5, 7, 8, 9, 10, 11

(UNM &LBERI),

12/13

(UNM &LBERI),

14,

17, 18, 19, 21

(UNM &LBERI),

26, 27, 28, 29

(UNM &LBERI),

35

(UNM/ASU),

36, 49, 50, 51, 52, 53, 55, 56, and 57

.

2. Estimates of subcontractors expenses from prior month if subcontractor did not submit a bill. List for each subcontractor. If subcontractors were not working or did not incur any cost in current or prior month, then include a statement to this effect.

LBERI, Cerus, ASU and UTSA all submitted invoices to UNM in the prior month. The UNM invoice to

NIAID, being submitted on 4/15/09, will include subcontractor invoices from ASU, LBERI and Cerus.

Though UTSA submitted an invoice to UNM at the end of March, UNM did not pay the invoice before the financial close of March.

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Period: 10/01/2008 to 3/31/2009

Due Date: 4/7/2009



To date, administrative costs have been billed to NIAID that are associated with Milestones 1 and 2 and with the management of the scientific milestones.

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