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CHIMERIC CD40L/SHIV VLPs ENHANCE DENDRITIC CELL ACTIVATION AND BOOST IMMUNE RESPONSES AGAINST HIV

CHIMERIC CD40L/SHIV VLPs ENHANCE DENDRITIC CELL ACTIVATION AND BOOST IMMUNE RESPONSES AGAINST HIV. Qizhi Cathy Yao, M.D., Ph.D. Molecular Surgeon Research Center Michael E. DeBakey Department of Surgery Baylor College of Medicine. Production of Chimeric CD40L/SHIV VLPs. rBV-HIVEnv.

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CHIMERIC CD40L/SHIV VLPs ENHANCE DENDRITIC CELL ACTIVATION AND BOOST IMMUNE RESPONSES AGAINST HIV

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  1. CHIMERIC CD40L/SHIV VLPs ENHANCE DENDRITIC CELL ACTIVATION AND BOOST IMMUNE RESPONSES AGAINST HIV Qizhi Cathy Yao, M.D., Ph.D. Molecular Surgeon Research Center Michael E. DeBakey Department of Surgery Baylor College of Medicine

  2. Production of Chimeric CD40L/SHIV VLPs rBV-HIVEnv rBV-SIVGag rBV-hCD40L Env. hCD40L CD40L/SHIV VLP Sf-9 cells

  3. B. A. Sf9 + rBV-hCD40L Sf9 + rBV-mCD40L 50 50 40 40 30 30 75.0% 87.2% Counts Counts 20 20 M1 M1 10 10 0 0 0 0 2 2 3 3 1 1 4 4 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H Surface expression of hCD40L and mCD40L

  4. C. 500 ng VLPs 1000 ng VLPs 1800 1600 1400 1200 1000 hCD40L Levels in VLPs (pg) 800 600 400 200 0 SHIV-VLP hCD40L/SHIV-VLP incorporation of hCD40L in chimeric hCD40L/SHIV-VLPs

  5. D. 500 ng VLPs 2000 1000 ng VLPs 1800 1600 1400 1200 mCD40L Levels in VLPs (pg) 1000 800 600 400 200 0 SHIV-VLP mCD40L/SHIV-VLP incorporation of mCD40L in chimeric mCD40L/SHIV-VLPs

  6. B. A. iDC+SHIV-VLP iDC+hCD40L/SHIV-VLP 140 140 120 120 100 100 80 Counts Counts 80 60 60 51.2% 62.1% 40 40 M1 M1 20 20 0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL1-Height FL1-Height Enhanced binding of SHIV-VLPs with DCs

  7. C. CD40 CD83 CD86 1.6% 91.2% 31.2% Untreated 92.5% 82.3% 43.8% LPS 42.9% 94.5% 80.3% SHIV-VLP 72.1% 95.8% 87.6% hCD40L/SHIV-VLP 6.9% 93.1% 41.5% Heated hCD40L/SHIV-VLP Enhanced phenotypic activation of DCs by chimeric hCD40L/SHIV-VLPs

  8. D. 1 5 Untreated SHIV-VLP+hCD40L 2 6 SHIV-VLP hCD40L Ab+hCD40L/SHIV-VLP 3 7 hCD40L/SHIV-VLP Irr. Ab+hCD40L/SHIV-VLP 100 4 8 hCD40L LPS 80 60 Positive Cells (%) 40 20 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 CD40 CD83 Blockade of hCD40L/SHIV-VLPs-induced DC activation by hCD40L specific antibody

  9. 1 5 Untreated SHIV-VLP+hCD40L 2 6 SHIV-VLP hCD40L Ab+hCD40L/SHIV-VLP 3 7 hCD40L/SHIV-VLP Irr. Ab+hCD40L/SHIV-VLP 4 8 hCD40L LPS 250 200 150 CD86 MFI 100 50 0 1 2 3 4 5 6 7 8

  10. Elevated allogeneic T-cell-stimulatory activity of chimeric CD40L/SHIV-VLPs-activated DCs 1. Untreated 4. hCD40L/SHIV-VLP 5. Heated hCD40L/SHIV-VLP 2. LPS 3. SHIV-VLP 100000 ** 80000 * 60000 [3H] Thymidine (CPM) 40000 20000 0 1 2 3 4 5

  11. Blockade of allogeneic T-cell-stimulatory activity ofhCD40L/SHIV-VLPs-induced DC by hCD40L Ab hCD40L/SHIV-VLP hCD40L+SHIV-VLP hCD40L Ab+hCD40L/SHIV-VLP 80000 irr. Ab+hCD40L/SHIV-VLP * 60000 [3H] Thymidine (CPM) 40000 20000 1:40 1:80 1:160 DC and T Cell Ratio

  12. Effect of hCD40L antibody on hCD40L/SHIV-VLP-induced secretion of Th1 type of cytokines.

  13. Elevated anti-HIV Env Ab production in chimeric mCD40L/SHIV-VLP immunized mice SIV-VLP SHIV-VLP 0.16 mCD40L/SHIV-VLP * 0.12 Anti-HIV Env IgG (O.D.405 nm) 0.08 0.04 0 20 μg VLPs 100 μg VLPs

  14. Elevated levels of IFN-γproduction in mCD40L/SIV-VLPs immunized mouse splenocytes upon SIV Gag peptides pulse SIV-VLP mCD40L/SIV-VLP 1200 * 1000 800 * # of spots/106 splenocytes 600 400 200 0 Gag pool 1 Gag pool 2 IFN-γ

  15. Elevated levels of IL-4 production in mCD40L/SIV-VLPs immunized mouse splenocytes upon SIV Gag peptides pulse SIV-VLP mCD40L/SIV-VLP 350 300 * * 250 200 # of spots/106 splenocytes 150 100 50 0 Gag pool 1 Gag pool 2 IL-4

  16. Elevated cytokines production in mCD40L/SHIV-VLPs immunized mouse splenocytes stimulated with Gag or Env peptides SHIV-VLP (20 μg) mCD40L/SHIV-VLP (20 μg) SHIV-VLP (100 μg) mCD40L/SHIV-VLP (100 μg) 2500 * ** 2000 * 1500 * # of Spots/106 Splenocytes 1000 * 500 0 Gag pool 1 Gag pool 2 Env IFN-γ

  17. SHIV-VLP (20 μg) mCD40L/SHIV-VLP (20 μg) SHIV-VLP (100 μg) mCD40L/SHIV-VLP (100 μg) 1600 ** 1400 1200 * 1000 # of Spots/106 Splenocytes 800 600 400 200 0 Gag pool 1 Gag pool 2 Env IL-4

  18. 1.6 FL1-H 2.8 4.1 FL1-H 4.5 FL1-H 4 4 4 4 4 4 4 4 4 4 10 10 10 10 10 10 10 10 10 10 6.9 FL1-H 0 0 0 0 0 0 0 0 0 0 10 10 10 10 10 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 FL1-H HIV Env. SIV Gag peptides peptides 1.4 Group/PBS 0 1 2 3 4 10 10 10 10 10 3.1 FL1-H SHIV VLP 20 μg 0 1 2 3 4 10 10 10 10 10 4.4 FL1-H SHIV VLP 100 μg 4.7 FL1-H mCD40L/SHIV VLP 20 μg IFN-γ mCD40L/SHIV 7.4 FL1-H VLP 100 μg 0 1 2 3 10 10 10 10 FL1-H CD8

  19. Conclusions • Chimeric CD40L/SHIV VLPs can phenotypically and functionally activate DC in vitro. • Chimeric CD40L/SHIV VLPs induce strong humoral and cellular immune responses in a mouse model.

  20. Acknowledgments Yao’s Lab Dr. Rongxin Zhang Dr. Uddalak Bharadwaj Dr. David Doan Collaborators Dr. Min Li Dr. Johnny Chen

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