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Genetic Immunization with Recombinant Lentivector

This presentation by Dr. Yukai He explores the innovative use of recombinant lentivectors for genetic immunization. It discusses various types of vaccines, including conventional, therapeutic, and genetic vaccines, focusing on how lentivectors outperform others in inducing robust T-cell immunity, particularly CD8+ T cells. The talk outlines methodologies for ex vivo and in vivo immunization approaches, highlights the significance of antigen presentation, and compares the efficacy of different immunization strategies while addressing immune responses against chronic infections and tumors.

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Genetic Immunization with Recombinant Lentivector

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  1. Genetic Immunization with Recombinant Lentivector Yukai He, MD/PhD Assistant Professor Departments of Dermatology and Immunology, University of Pittsburgh, School of Medicine

  2. Salk and Youngner At University of Pittsburgh Vaccines • Conventional vaccines • Attenuated organisms: Oral Polio vaccine (Sabin vaccine) • Inactivated organisms: Injected polio vaccine (Salk Polio vaccine) • Subunit protein vaccines: HBV vaccine Immune correlates: Neutralizing Ab B cell vaccines

  3. T cell vaccines • HIV, Malaria, TB, Tumor • Therapeutic vaccines: Chronic HBV infections Attenuated organisms Genetic vaccines DNA vaccine: Naked DNA, gene gun Viral vectors: Adenovector, alpha viral vector, vaccinia vector,lentivector, AAV

  4. Recombinant Lentivector

  5. -Globin-intron Globin-pA Gag-pol RRE CMV-p pRSV Rev HIV-pA pLP1 -Globin-intron Globin-pA VSV-G CMV-p  RRE pSV40 pRSV/5LTR TRIP EM7 Blasticidin U3/HIV 3’-LTR CMV-EGFP pLP2 SD SA Trip-EGFP pLP/VSV-G B: Transfer Plasmids 3rd Generation of Lentivector A: Packaging plasmids

  6. Adenoviral vector High titer High transduction efficiency Non-intergation Short term gene expression Long term Ag presentation Immune dominant antivector immunity Pre-existing antivector immunity Lentiviral vector High titer High transduction efficiency Intergration or Non-integration Long term gene expression and Ag presentation Low or no immune dominant antivector immunity No pre-existing antivector immunity Comparison between Adenovector and Lentivector

  7. T cell immunity elicted by lentivector immunization Ex vivo Approach In vivo Approach

  8. Ex vivo Approach (He et al., 2005, JI) • Prepare BMDCs and recombinant lentivector • Transduction of BMDCs ex vivo • Immunize mice with BMDCs • Monitor the T cell immunity by in vivo killing assay and intracellular staining of IFN • Examine the antitumor effect

  9. PBS-DC EGFP-lv-DC CD11c B7.2 PBS-DC EGFP-lv-DC GFP Transduction of BMDCs ex vivo by Lentivector

  10. MFI pg/ million cells IFN-gamma produced by allogenic T cells MLR 180 800 160 700 140 PBS-DC 600 120 OVA-lvv-DC 500 100 400 pg/ml cpm (X10-3) 80 300 PBS-DC 60 200 OVA-lvv-DC 40 100 20 0 0 1:50 1:100 1:200 1:400 1:50 1:100 1:200 1:400 1:800 1:1600 DC:responder ratios DC: responder ratios Lentivector does not change the intrinsic properties of transduced BMDCs

  11. Naive PBS-DC Pulsed DC Transduced DC 50.35 49.65 51.18 48.82 95.11 4.9 99.91 0.09 In vivo killing assay CFSE 0 per 100K 68 per 100K 493 per 100K 1895 per 100K CD8 IC-staining of IFN 4 per 100K 30 per 100K 160 per 100K 150 per 100K CD4 IFN- Lentivector Transduced BMDCs induce strong T cell immune responses

  12. (0/10) (0/10) Tumor area (mm) (10/10) Antitumor effect of lentivector mediated genetic immunization

  13. In vivo approach (He et al., 2006 Immunity) • Compare the efficacy of in vivo and ex vivo approach • Immunize mice with recombinant lentivector • Monitor T cell immunity • Examine the antitumor effect

  14. Lentivector induce strongest CD8 T cell immunity

  15. Lentivector induce persistent and potent CD8 T cell immunity

  16. Antitumor effect of lentivector mediated genetic immunization

  17. 49.94 50.10 21329 12 20491 8 CD8 CD8 CD8 119 144 IFN IFN IFN 27365 20 IM IM+Electroporation 48.06 51.98 91 DNA 49.51 50.49 HBsAg specific CTL activity following genetic immunization Naive

  18. HBS-lvv CD8 CD8 In vivo killing assay IFN IFN 27644 211 112 IC-staining IM S.C 29214 803 89.95 10.06 99.64 0.36 165

  19. Comparative efficacy of different immunization approaches

  20. Mechanism of T cell priming in lentivector mediate genetic immunization • He et al., 2006 Immunity

  21. 1 T T T T T T sDC Paradigm: Direct Priming Infection/Danger Signal 1: Resting sDC 2: Activated sDC sDC Ag Skin 2

  22. Tissue derived DCs: LC Dermal DCs Other tissue DCs Blood derived DCs: CD8+ CD4+ CD8-CD4- pDCs DC Network

  23. Heath and Carbone groups indicate that LCs are not directly involved with priming of naïve T cells after skin HSV infection (Allan et al, Science 2003; 301:1925) CD8+ LN resident DCs prime naïve T cells via cross priming after HSV, Influenza A, vaccinia virus, LCMV, and Listeria Monocytogene infection (Allan et al, Science 2003; Belz et al., 2004; Smith et al., 2003; Belz et al., 2005)

  24. 1. Resting sDC 2. Activated sDC 3.Resident CD8 DC 1 Ag T T T T T T Paradigm Shift: Cross Priming Infection/Danger Signal sDC Ag Skin 2 (Carbone et al., 2004; Heath et al., 2004; Serbina and Pamer, 2003)

  25. Questions raised • How to efficiently Ag transfer and faithfully transfer the environmental cues from sDCs to LN resident DCs • Is this a generalized truth or restricted to the few viruses studied? • Cytopathic virus or with well-described mechanism for immune evasion (Bosnjak et al., 2005; Engelmayer et al., 1999; Larsson et al., 2004; Sevilla et al., 2003). • Non-cytopathic vectors such as lentivector that was shown not interfere with the APC function of transfected DCs (He et al, 2005) are able to directly prime T cells.

  26. Luciferase activity RT-PCR DLN DC subsets By Cell sorting Co-culture with OT-I cells 2 days Luc-lvv OVA-lvv Analysis by Flow cytometry 3H incorporation 3 days Experimental Design

  27. OT-I alone 58.18% 96.95% 41.83% 3.09% CD11c+ cells CFSE B220 CD8-B220-DC CD8+B220-DC pDCs 95.76% 4.27% CD8 99.28% 0.74% CD11c+CD8-B220- prime naïve CD8 T cells

  28. CD11c+ Cells CD8-PE CD11b CD11c+CD8-CD11b+ prime naïve CD8 T cells

  29. CD11c+ Cells DEC205 CD8 CD11c+ Cells DEC205 CD8 CD11c+CD8-/loDEC205+ sDCs prime CD8 T cells LV VV

  30. CD11c+ Cells DEC205 CD8 CD11c+ Cells B220 CD8 Transgene expression is only found in sDC

  31. OVA-lvv 7 hours 7 hours 20 hours 20 hours 46 hours 46 hours DEC205+ DEC205+ DEC205+ DEC205+ DEC205+ DEC205+ CD8+ CD8+ CD8+ CD8+ CD8+ CD8+ OVA S15 OVA-VV OVA S15

  32. Day 1-4 Day 2-5 Day 5-8 Day 12-15 Day 21-24 91.81% 8.19% 73.13% 27.09% 94.71% 5.34% 56.65% 43.38% 88.88% 11.20% 95.31% 4.73% 46.74% 53.56% 34.90% 65.32% 2.69% 97.35% 92.29% 7.71% Immunization with lentivector showed prolonged in vivo Ag presentation

  33. 175 491 856 Naïve FITC FITC + PT CD11c-PE FITC Inhibition of skin DC migration by injecting pertusis toxin FITC+ DCs in the DLN migrate from skin

  34. CD11c FITC FITC- FITC+ CD11c CD11c FITC FITC DEC205 DEC205 CD8 CD8

  35. Summary: Paradigm Found • 1.In contrast to previous studies using HSV, IAV, and VV, skin derived DCs appear to play a dominant role in priming naïve T cells after LV immunization. • Classical Paradigm is restored in this LV mediated immunization system • Immunization with non-cytopathic LV result in potent effector and memory CD8 T cell responses underscored by extended time of direct Ag presentation. • It remains to be determined if LC, DDC, or both play the of APC after LV immunization

  36. Acknowledgements: Jiying Zhang Cara Donahue Louis D. Falo, Jr., MD, PhD Chairman of the Department of Dermatology University of Pittsburgh Dr. Jonathan Yewdell of NIH for providing VV-OVA This research is supported the grant from NIAMS to Dr. Yukai He.

  37. Introduction to Lentivector • T cell immune responses induced by lentivector immunization • Mechanism of lentivector mediated genetic immunization

  38. Lentivector is less immunogenic in inducing antivector immunity

  39. Elicitation of potent effector and memory CD8 T cell response

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