HIV Infection and Human Blood Cell Development

1. HIV Infection and Human Blood Cell Development Lessons Learned from the Humanized Mouse

2. Use of humanized mouse model as a translational medicine tool • Past successes: • Pathogenesis studies • Drug screening • Gene therapy • Future aims: • Gene therapy • Vaccine development • Immune reconstitution/enhancement • Viral transmission • Viral latency/ eradication • Organ regeneration/replacement • Novel therapeutic development

3. Immunodeficient Mice SCID, NSG Rag2-/- Black 6 SCID

7. Use of Humanized micein HIV-related studies

10. Model #1: The hu-PBL SCID Mouse Model (the Mosier Model) • First humanized mouse used in HIV studies. • Involves injecting human Peripheral Blood Lymphocytes into the peritoneum of SCID mice. Cells are typically removed from mice by intraperitoneal lavage. • Key Strength: can assess effects rapidly and directly on mature human blood cells in vivo. • Key Weaknesses: limited number of time points, short experimental duration (<3-4 weeks).

11. Typical Experiment: hu-PBL SCID Mouse Model Cells HIV Drug 7 -14 days 7 -21 days Lavage and Assessment: Real Time Quantitative PCR Typically there are 6-30 mice per experiment. The limiting factor in these studies is the number of human cells. You need >2 x 107 human cells/mouse.

12. Analysis of radio-labeled HIV specific antibody 213Bismuth HIV Infected 213Bismuth HIV Infected

13. 213Bismuth 213Bismuth

14. Experimental Results n = 5 mice/group Dadachova et al. PLoS One 2012

15. Conclusions: anti gp41 antibody in hu-PBL SCID mice • Bismuth213- labeled neutralizing anti-gp41 monoclonal antibody targets and eliminates HIV infected cells in vivo. • Platelet counts and Pathology similar in all experimental conditions: • Low toxicity of radioactive compound • Eliminates productively infected cells. • Decreases Viral DNA load.

16. Model #2: The SCID-hu thy/liv mouse model • Developed at Stanford, the SCID-human fetal thymus and liver model. • Involves transplanting human fetal thymus and liver in SCID mice. Cells or HIV is then injected into tissue. Tissue is obtained by biopsy. • Key Strengths: can assess viral infection of human T cells in human tissue in vivo, system to study T cell development. • Key Weaknesses: surgery required, technically complex.

17. The SCID-hu Mouse Model • Established in the late 1980’s/early 1990’s as a model system to study HIV pathogenesis in vivo. • Played a key role in studies on: • HIV Pathogenesis • Gene Therapy • HIV Latency • Embryonic Stem Cell Development • Engineering T cell Immunity

18. The thymus is the organ that generates T cells How does HIV infection perturb T cell development?

19. Thymopoiesis Peripheral Circulation Thymus CD4 SP Quiescent CD4+ / CD45RA+ CD4+ / CD8+ Transcriptionally Active CD4- / CD8- CD8+ / CD45RA+ CD8 SP Quiescent

20. The SCID-hu mouse model Human fetal liver Human fetal thymus Thy/Liv implant 3-4 months CD8 SCID-hu mouse

22. Typical Experiment: hu-PBL SCID Mouse Model HIV Biopsy and Assessment SCID-hu SCID-hu Tissue processing 3-22 weeks • Assay • PCR • Flow • cytometry Experiments typically consist of 6-30 mice, and have 3 time points. The limiting factor is the amount of fetal tissue.

23. Uninfected HIV infected

25. HIV infection causes loss of immature thymocytesWhat viral factors are involved in this process?

27. HIV Reporter Virus muHSA (CD24)

28. HSA Expression in Thymocytes Jamieson et al.

29. What is the effect of HIV infection on the thymic microenvironment? Do high levels of HIV destroy the ability of thymic stroma to direct T cell differentiation?

32. Why is reconstitution of thymocytes transient?

33. Viral / Thymocyte Dynamics Following Antiretroviral Therapy

34. Conclusions • Reconstitution of thymopoiesis is transient following HAART. • The transiency is caused by breakthrough in viral replication to antiretroviral treatment. • The SCID-hu thy/liv model is highly useful in examining HIV infection in the context of developing T cells.

35. Modeling HIV Latency

36. HIV-1 CD4 SP /HSA - 4-6 weeks Biopsy + Protease Inhibitor CD8 SP /HSA - Thy/Liv Implant

37. Day 3 21% MFI: 510 CD24 79% CD3 / CD28 Co-Stimulation CD45 Unstimulated 5% MFI: 225 CD24 95% CD45 Latent HIV in Thymocytes from SCID-hu Mice Day 0 99% <1% CD4 + Protease Inhibitor CD24 <1% 99% CD8

38. Targeting The Latent Reservoir

39. The Search for Agents That Activate Latent HIV Prostratin Phorbol ester Used in tea in Samoa to treat various illnesses Activates latent virus without inducing T cell replication Further testing is required to define effects on immune system IL-7 Naturally occuring cytokine Induces some cell proliferation, but phenotype is maintained Potently induces expression of latent HIV Further development is required

40. Infected Cell gp120 Pseudomonas Exotoxin Anti-HIV Immunotoxin Anti-gp120 3B3:N31H/Q100eY(dsFv)-PE McHugh et al.; 2002

41. Elimination of Latent HIV

42. Conclusions • Immunotoxins can be used to kill cells induced to express previously latent virus • Pre-treatment with IL-7 or with prostratin plus immunotoxin results in a decrease in rescuable latent virus upon subsequent co-stimulation. • These agents may prove useful as adjunctive therapeutics to purge the latent HIV reservoir

43. Model #3: The Non-obese diabetic (NOD), SCID, IL-2 receptor γ knockout (NSG), humanized bone marrow, fetal liver and thymus (BLT) mouse modelThe NSG-BLT model • Recently developed model, pioneered by J. Victor Garcia in Texas. • Involves transplanting human fetal thymus and liver in NSG mice, the irradiating them and the injecting human stem cells intraveneously, which allow them to become engrafted in mouse bone marrow. Mice become engrafted with multiple human cell types that arise from stem cells within 6-8 weeks. • HIV is then injected. HIV replication and peripheral blood cells are monitored following bleeding of the mice. Tissue is obtained by biopsy and/ or sacrificing mice.

44. Model #3: The Non-obese diabetic, SCID, IL-2 receptor γ knockout (NSG), humanized bone marrow, fetal liver and thymus (BLT) mouse model • Key Strengths: can assess viral infection of multiple types of human cells in vivo, slow and steady rate of T cell depletion and viral replication (mimics natural history in humans), easy to manipulate, develop immune responses. • Key Weaknesses: surgery required, technically complex, immuodeficient status of mice make them highly susceptible to graft versus host disease, lower experimental numbers.

45. Humanized Mouse Model of HIV Infection: The NSG-BLT Model CD34+ CD34+ CD34+ CD34+ CD34+ 1. Implant fetal thymus and liver tissue. Infect with HIV-1 thy liv Irradiate 6-12 weeks 3 weeks 4. Analyze human cell reconstitution 3. i.v. Inject 2. Sort CD34+ Stem Cells 5. Analyze effects of infection NSG NSG Each experiment typically contains 6-15 mice. Human cell reconstitution frequency is far lower than SCID –hu.

46. Multlineage Hematopoesis in Humanized mice CD34+ Myeloid stem cell Lymphoid stem cell BM Stem cell B progenitor Basophil progenitor T progenitor Eosinophil progenitor Myelomonocytic progenitor Erythroid progenitor Megakaryoblast DP Thymocyte Monocyte NK Cell Megakaryocyte CD8+ T cell CD4+ T cell Red blood cells Platelets Eosinophil Basophil Neutrophil B cell Macrophage

47. Multilineage Hematopoiesis in NSG-LTL mice %CD45+ mean=53% ± 29% range 19%-80% n=12

48. HIV Infection of NSG-BLT mice • Mice were either • Untreated • HIV infected • HIV infected but treated • with pre-exposure prophylaxis • of emtricitabine (FTC)/ • tenofovirdisoproxil • fumarate (TDF) Conclusion: Pre-exposure prophylaxis Prevents infection in this model Denton et al.,PLoS Med. 2008 Jan 15;5(1)

49.  Engineering HIV-Specific T-Cell Immunity

50. Targeting immune responses could augment rejection of Infectious agents (chronic viruses) or tumors in certain individuals or disease states HIV disease: weakened immune system, viral drift Cancer: Escape of tumor cells from immune surveillance Transgenic mouse models suggest that introduction of antigen receptors into stem cells can result in functional effector cells targeting the antigen Can this type of approach be done in humans? Can we enhance immune capabilities in humans?