1. Models Systems to Study HIV Emily L. Lowe, Ph.D. Microbiology, Immunology and Molecular Genetics UCLA

2. Models Systems to Study HIV: In vitro and In vivo In vitro: Cell lines, primary cells In vivo: Humanized mice In vivo: Non-human Primates

3. Models Systems to Study HIV: In vitro and In vivo Genetically modified viruses – Modify the envelope to allow entry into more (or less) cell types – Add a fluorescent protein to enable better visualization Green fluorescent protein (GFP), green mCherry, red SIV (Simian Immunodeficiency Virus) SHIV: chimeras of HIV and SIV Yu et al. HIV traffics through a specialized, surface-accessible intracellular compartment during trans-infection of T cells by mature dendritic cells. PLoS Pathog. 4 (8). 2008. e1000134.

4. In vitro Models to Study HIV In vitro: Taking place in a test tube, culture dish or elsewhere OUTSIDE a living organism Excellent tools for simple questions with limited variables – Best for testing more focused approaches Cells are easily manipulated Cost efficient Scalable Excellent for initial drug screens

5. Types of In vitro Models Cancer cell lines are IMMORTALIZED – Can be expanded in culture for long periods of time – Are easily manipulated to express (or not) CD4, CCR5 and/or CXCR4 as well as host restriction factors “Primary cells” come from people or animal subjects – Can purified to look at single or multiple populations – Can only be maintained in culture for short periods – Can be manipulated but not as easily as cancer cell lines

6. Examples of in vitro applications To see HIV virions To see an HIV infected cell infect another cell To see where HIV goes in a cell To see what HIV interacts with in a cell Identify latency inducers

7. In vitro study to see HIV virions June et al., Direct Visualization of HIV-with Correlative Live-Cell Microscopy and Cry-Electron Tomography. Structure. 19 (11). 2011. 1573-1581. Cryofluorescence Light Microscope 170X3500X50,000X

8. June et al., Direct Visualization of HIV-with Correlative Live-Cell Microscopy and Cry-Electron Tomography. Structure. 19 (11). 2011. 1573-1581. In vitro study to see HIV virions

9. In vitro study to see an HIV infected cell infect another cell Dr. Thomas Huser’s Lab http://cbst.ucdavis.edu/research/hiv-transmission-visualization

10. In vitro study to see where HIV goes in a cell it has infected Campbell et al., Visualization of a proteasome-independent intermediate during restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561

11. In vitro study to see where HIV goes in a cell it has infected Green = HIV Red = “body” Campbell et al., Visualization of a proteasome-independent intermediate during restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561

12. In vitro study to see what HIV interacts with inside a cell Campbell et al., Visualization of a proteasome-independent intermediate during restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561 Green = HIV Red = host restriction factor Orange = interaction!!!

13. In vitro study to identify latency inducers Kim et al., Recruitment of THIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency. EMBO J. 25. 2006. 3596-3604.

14. In vitro studies have given us a lot but… One major limitation to vaccine and therapeutic cure research has been the lack of an animal model that recapitulates all of the salient features of HIV-1 infection in humans. In vivo: Experimentation using a whole, living organism

15. In vivo Models to Study HIV When considering species other than human as models for HIV-1 infection, the cellular proteins of the species must support viral replication. Humanized mice Non-human primates

16. In vivo Models to Study HIV: Humanized Mice Most importantly, these animals CAN be infected with HIV! Develop systemic viremia CD4 T cell loss

17. Akkina, R. New generation humanized mice for virus research: comparative aspects and future prospects. Virology. 435 (1). 2013. 14-28.

18. In vivo Models to Study HIV: Humanized Mice Stem cells can be genetically manipulated (gene therapy) Not terribly cost effective – Surgery requires technical skill – Cannot be bred – Special housing/handling due to immune compromised status Scalable allows for multiple “n” – Can make up to 30 mice per tissue pair – Excellent for initial drug-toxicity/efficacy screens IV and mucosal routes of infection possible Non-hematopoietic cells (lung, intestine mucosa) cannot be studied in organ systems

19. In vivo Models to Study HIV: Non-human primates Many species of African monkeys and apes are natural hosts for SIV, but generally do not develop disease as a consequence of infection.

20. ©2011 by Cold Spring Harbor Laboratory Press Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 Natural Hosts (African) African green monkey SIVagm

21. In vivo Models to Study HIV: Non-human primates By contrast, infection of Asian macaques, which are NOT natural hosts for primate lentiviruses, with certain strains of SIV results in high viral loads, progressive CD4+ T cell depletion and opportunistic infections.

22. ©2011 by Cold Spring Harbor Laboratory Press Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 Natural Hosts (African) Non-natural Hosts (Asian)

23. In vivo Models to Study HIV: Non-human primates and SIV HIV-1 group M, which is the most prevalent HIV strain, jumped from chimpanzees into humans. http://www.awf.org/content/wildlife/detail/chimpanzee HIV-2 originated in sooty mangabeys and is responsible for fewer infections than HIV-1 http://pin.primate.wisc.edu/factsh eets/entry/sooty_mangabey SIV cpz HIV-1 SIV smm HIV-2SIV mac

24. In vivo Models to Study HIV: Non-human primates and SIV SIV: Simian Immunodeficieny Virus – First isolated at the New England Primate Research Center, Massachusetts, from rhesus macaques with a transmissible form of immunodeficiency characterized by opportunistic infections and tumours – Later traced to an outbreak of lymphoma in the 1970s among macaques that were housed at the California National Primate Research Center, California – Monkeys might have received tissues from SIV- infected sooty mangabeys during experiments aiming to develop a non-human primate model for prion disease

25. In vivo Models to Study HIV: Non-human primates and SHIVs SHIV – Simian immunodeficieny virus containing HIV sequences/elements – HIV-based vaccines cannot be tested with SIV – SIV is not sensitive to many drugs that inhibit HIV-1 – SIV uses CD4 and CCR5 (like HIV-1) but my also use other co-receptors complicating testing of entry inhibitors Env-SHIV RT-SHIV stHIV-1

26. In vivo Models to Study HIV: Non-human primates and SHIVs Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

27. In vivo Models to Study HIV: Non-human primates and SHIVs Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

28. In vivo Models to Study HIV: Non-human primates and SHIVs Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867. Pig tailed macaque

29. Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867. In vivo Models to Study HIV: Non-human primates

30. Most commonly used Pathogenesis similar to HIV-1 High viral loads Progressive depletion of mucosal or peripheral CD4+ T cells Destruction of lymph node architecture Progress AIDS faster (1-2 yrs) Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

31. Next most commonly used Lower viral loads and less CD4+ T cell depletion Progress AIDS faster (within 42 weeks) Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

32. Least used SIV strains are less pathogenic (probably because they are passaged in Rhesus macaques)

33. In vivo Models to Study HIV: Non-human primates Stem cells can be genetically manipulated (gene therapy) Cost prohibitive Primate research contains ethical controvery Not scalable IV and mucosal routes of infection possible All tissues match (non-hematopoietic and immune) allowing for organ systems to be studied

34. Models to study HIV Cats and Feline Immunodeficiency Virus Wongsrikeao et al., Antiviral restriction factor transgenesis in the domestic cat. Nature Methods. 8. 2011. 853-859.

35. Thank you!