Genetic Immunization with Recombinant Lentivector

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

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

24. 1 T T T T T T sDC Paradigm: Direct Priming Infection/Danger Signal 1: Resting sDC 2: Activated sDC sDC Ag Skin 2

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

26. 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)

27. 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)

28. 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.

29. 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

30. 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

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

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

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

34. 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

35. 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

36. 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

37. CD11c FITC FITC- FITC+ CD11c CD11c FITC FITC DEC205 DEC205 CD8 CD8

38. 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

39. 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.

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

41. Lentivector is less immunogenic in inducing antivector immunity

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