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1 Progress Report 2/04/2009 TVDC team – UNM Prepared by Terry Wu & Amanda DuBois.

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Presentation on theme: "1 Progress Report 2/04/2009 TVDC team – UNM Prepared by Terry Wu & Amanda DuBois."— Presentation transcript:

1 1 Progress Report 2/04/2009 TVDC team – UNM Prepared by Terry Wu & Amanda DuBois

2 2 Active Milestones Active Milestones: 5, 11, 12/13, 14, 17, 18, 19, 21, 29, 35 Today’s presentation will include: 5. Effect of LVS vaccination dose on protection of Fischer rats against i.t. SCHU S4 challenge 12/13.ELISA to titer vaccinated human serum 17.Repeat of SCHU S4 growth kinetics in passively immunized Fischer 344 rats 21.a) SCHU S4 infection of human PBMC b) Multifunctional T cells in NHP 29. Plans for screening Ft peptide library from ASU

3 3 Milestone 21: Multifunctional T cell assay T cell responses from vaccinated animals Develop assay to detect CD4+ multifunctional T cells Induction of multifunctional T cells after LVS vaccination Mice (UNM) Non-human primates (LBERI/UNM) Blue: Steps in the milestone Red: Completed Green: In progress Rats (UNM) Recruitment of multifunctional cells after Schu S4 challenge Induction of multifunctional T cells after LVS vaccination Recruitment of multifunctional cells after Schu S4 challenge Develop assay to detect CD4+ multifunctional T cells Induction of multifunctional T cells after LVS vaccination Induction of multifunctional cells after respiratory LVS challenge Recruitment of multifunctional cells after Schu S4 challenge Develop assay to detect CD4+ multifunctional T cells

4 4 Multiparameter flow approach TNFα-A488IL-2-PEIFNγ-PE*Cy7 IL-2-PE TNF α -A488 IL-2-PE TNF α -A488

5 Two doses of HK-LVS stimulated a high frequency of CD4+ lung cells producing TNF , IL-2, and IFN  5

6 %CD4+ lung cells secreting TNF , IL-2, and IFN  (Uninfected vs LVS treated NHP) 6 Uninfected NHP (fresh) LVS treated NHP (Fresh) LVS treated NHP (Frozen)

7 Animal A00868 Cynomolgus macaque vaccinated by subcutaneous inoculation with 2.7x10 6 cfu LVS on 11/29/06 Inoculated by bronchoscope instillation with 1x10 5 cfu LVS on 1/9/2009 Day 12 after boost/infection: samples of blood, lungs, tracheobronchial lymph nodes (TBLN), bronchoalveolar lavage (BAL), liver, and spleen collected 7

8 Measured outcomes (data not yet available) Cellular recruitment to the site of infection: –phenotyping of blood and single cell suspensions isolated from lungs, TBLN, and BAL via FACS analysis –differential staining of cytospins prepared from single cell suspensions of lungs, TBLN, and BAL Cytokine and chemokine response to infection: –Cytokine and chemokine levels in supernatant from freshly isolated, homogenized lung, TBLN, spleen and liver tissue, as well as in BAL fluid –Cytokine and chemokine levels in supernatant from single cell suspensions from lungs, TBLN, and BAL cultured overnight in the presence of HK-LVS or mitogenic stimuli 8

9 Frequency of multifunctional cells in lungs following stimulation with PMA/ionomycin and HK-LVS 99 Uninfected NHP (frozen) LVS treated NHP (Fresh)

10 10 Uninfected NHP (frozen) LVS vaccinated/LVS boosted NHP (Fresh) Pulmonary LVS NHP (Frozen) Low but detectable population of multifunctional cells responsive to HK-LVS in spleen of LVS vaccinated/LVS boosted NHP

11 Response to HK-LVS undetectable in TBLN from untreated or LVS vaccinated/LVS boosted NHP 11 Uninfected NHP (frozen) LVS treated NHP (Fresh) Only organ with detectable bacteria (~300 cfu in total TBLN)

12 Observations Stimulation with HK-LVS induced robust and slight increases in multifunctional cells present in lung and splenocyte populations, respectively, from LVS vaccinated/LVS boosted NHP –No increase in multifunctional cells from TBLN Treatment with PMA+Ionomycin was able to induce robust expression of all 3 cytokines and large increases in the frequency of multifunctional T cells in frozen “naive” and fresh LVS vaccinated/LVS boosted cells –Exception: fresh lung cells did not respond to PMA+Ionomycin 12

13 13 Milestone 21: Ongoing and upcoming experiments Cellular recruitment and cytokine/chemokine response data Presence of multifunctional cells in the lungs, TBLN, and spleen of LVS vaccinated/SchuS4 aerosol challenge survivors (Early March) Frequency of multifunctional cells in lungs, TBLN, and spleen of LVS vaccinated NHP before and after pulmonary SchuS4 infection

14 14 Milestone 5 – flow diagram Small animal models Fischer 344 rats BALB/c mice Guinea pigs SCHU S4 LD50 i.n. (i.t.) LVS LD50 s.c., i.d. i.n. LVS vaccination Clearance of vaccination strain SCHU S4 challenge (in/it) Clinical signs and survival Model selection SCHU S4 LD50 i.n. (i.t.) LVS LD50 s.c., i.d. i.n. LVS vaccination Clearance of vaccination strain SCHU S4 challenge (in/it) Clinical signs and survival SCHU S4 LD50 i.n. (i.t.) LVS LD50 s.c., i.d. i.n. LVS vaccination Clearance of vaccination strain SCHU S4 challenge (in/it) Clinical signs and survival Blue: Steps in the milestone Red: Completed Green: In progress Statistical analyses Robustness of Fischer 344 rat model LVS dose response Comparison of histopathology mice, rats, NHP

15 Protection Dependent on Vaccine Dose 15

16 Milestone 5: Plans Reduce LVS vaccination dose Complete histological analyses of tissues from SCHU S4-infected mouse, rat and NHP 16

17 17 Milestone 12/13 – flow diagram Assays for Detecting Relevant Immune Responses in Animals and Humans Mice (UNM)Humans (UNM) Blue: Steps in the milestone Red: Completed Green: In progress Rats (UNM) Generate reagents T cell IFNg production Determine and optimize assay sensitivity ELISA for Ab titer T cell proliferation Generate reagents T cell IFNg production Determine and optimize assay sensitivity ELISA for Ab titer T cell proliferation Generate reagents T cell IFNg production Determine and optimize assay sensitivity ELISA for Ab titer T cell proliferation

18 Anti-LVS Titer in Vaccinated Humans 18

19 Milestone 12/13: Plans Obtain sera from additional vaccinated individuals for standard pool? IFN  ELISpot with vaccinated human PBMC 19

20 20 MS 17: Characterization of Fischer 344 Rat Fischer 344 rats Humoral immunity Cell mediated immunity. LVS vaccination Passive transfer of serum Protection against i.t SCHU challenge Blue: Steps in the milestone Red: Completed Green: In progress Production of ascites fluid for CD4 and CD8 depletion In vivo depletion Pretection against i.t. SCHU SCHU challenge Active vs passive immunizatioin Purchase and culture hybridoma cell lines

21 21 Kinetics of SCHU S4 Growth & Dissemination in Passively-immunized Rats SpleenLiverLungs

22 22 Kinetics of SCHU S4 Growth & Dissemination in Passively-immunized Rats - Repeat

23 Milestone 17: Plans Complete bacterial burden experiment 23

24 24 MS 21: Assays in Vaccinated Humans Assay to measure activation of macrophage killing mechanisms in humans Macrophage T cells Determine the approximate yield of macrophages from whole blood (1-200 ml max) Determine and optimize cell number and MOI Positive control w/ IFN  Blue: Steps in the milestone Red: Completed Green: In progress Determine the approximate yield of PBMC and T cells from whole blood Test PBMC Test purified T cells in vitro expansion? Repeat w/ human vaccinee Monocyte-derived macrophages MonocytesPBMC Immu Naive

25 Proliferation of SCHU S4 in Human Monocytes 25

26 Infection of Human PBMC & Monocytes 26

27 Activation of PBMC and Monocytes with recombinant IFN  27

28 Milestone 21: Plans Determine whether human PBMC/monocyte effector activity can be increased beyond the level observed after IFN , eg by preactivation Addition of vaccinated human T cells 28

29 29 MS29: Plans Vaccinated NHP with LVS ASU prepared and shipped peptide array – 2065 linear expression element constructs UNM – Ordered needed reagents and materials Schedule – Feb 11 boost with LVS by bronchoscopy – Feb 22 assay with Lymph node cells and splenocytes – 7 peptides/well in duplicates, 16 plates total – IFN  ELIspot with cells and ELISA on culture supe

30 Action Items Julie Hutt: will convey the quality of the veterinary pathology slides that return to UNM from the North Carolina based vendor. Terry will send the sera titration figure to Freyja for review and will include in the 2/15/09 monthly tech report. Terry will copy the email to Barbara. (completed 2/5/09) Terry will purchased pooled normal human sera from a commercial vendor, if available Freyja: will get microagglutination protocol from Marcelo and send it to UNM; NIAID will make a batch of antigens (normal and O mutants) to share with DVC and UNM. Freyja: will advise Barbara on which Cerus lab bench protocols to move into “gold standard format”, in association with Cerus MS#40 MSCR. 30

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