Martha Lewis Blum, M.D. Ph.D. August 20, 2013

1. Curing HIV Martha Lewis Blum, M.D. Ph.D. August 20, 2013

2. Outline 1. ) Overview of HIV life cycle: Why is HIV so hard to eradicate? (2 slides) 2.) Review of current treatments for HIV: What is HAART, why can’t it cure HIV? (11 slides) 3.) Review of highly publicized “Berlin Patient” (2 slides) 4.) Overview of current strategies to eradicate HIV: “shock and kill” and stem cell therapies (2 slides)

3. HIV Life Cycle Virion binds CD4, then chemokine receptor CCR5,then fuses with cell. Core with genome inside is released into the cell. RT takes place in the cytoplasm. DNA copy is transported into the nucleus. Viral DNA is integrated into host cell chromosome by integrase. Viral DNA serves as template for making new virus proteins. New virus proteins are packaged into new virions and released from the cell. Virion matures after release and goes on to infect a new cell.

4. Anti-HIV drugs target multiple stages of the viral life cycle Attachment Entry Reverse transciption Integration Protein processing From: The design of drugs for HIV and HCV Erik De Clercq Nature Reviews Drug Discovery 6, 1001-1018 (December 2007)

5. Currently Licensed Anti-HIV Drugs 3TC lamivudine Epivir ABC abacavir Ziagen AZT or ZDVzidovudine Retrovir March 1987- first d4T stavudine Zerit ddI didanosine Videx EC FTC emtricitabineEmtriva TDF tenofovir Viread DLV delavirdine Rescriptor EFV efavirenz Sustiva (US) ETR etravirine Intelence NVP nevirapine Viramune rilpivirine Edurant APV amprenavir Agenerase FOS-APV fosamprenavirLexiva (US) ATV atazanavir Reyataz DRV darunavir Prezista IDV indinavir Crixivan LPV/RTV lopinavir + ritonavirKaletra NFV nelfinavir Viracept RTV ritonavir Norvir SQV saquinavir Invirase TPV tipranavir Aptivus T-20 enfuvirtide Fuzeon MVC maraviroc Selzentry (US) RAL raltegravir Isentress dolutegravir Trivicay August 2013-most recent NRTIs RT inhibitors NNRTIs Protease inhibitors Fusion inhibitor Attachment inhibitor Integrase inhibitor

6. Nucleoside RT inhibitors - NRTIs The first class of anti-HIV drugs developed, AZT for the first in this class (1987). All of the drugs in this class work by binding at the RT active site. They are incorporated into the growing DNA chain and cause chain termination. AZT is a thymidine analog, other drugs in this class are analogs of the other nucleosides.

7. Non-nucleoside RT inhibitors - NNRTIs This class is more structurally diverse, but all drugs in this class work by blocking RT at some other location than the active site. Binding of these agents causes conformational changes in the protein rendering the active site inaccessible. Active site NNRTI binding site

8. Protease Inhibitors - PIs (1995) These agents inhibit the viral protease encoded by the pol gene. This protease cleaves the Gag polyprotein, a final step required in the production of fully mature core structures required for infectious viral particles. From: HIV chemotherapy Douglas D. Richman Nature 410, 995-1001(19 April 2001)

9. The HAART Era Highly Active Anti-Retroviral Therapy. With the development of protease inhibitors it was then possible to target multiple stages of viral replication. Protease inhibitors were more potent/complete inhibitors of viral replication than RT inhibitors. The use of combination therapy with drugs to target RT and protease resulted in viral replication being reduced to undetectable levels for a sustained period of time.

10. Soon after the discovery of HIV it was determined that CD4 on the surface of T cells was necessary (but not sufficient) for viral entry. However, it wasn’t until early 1996 that it was determined that a chemokine receptor was required for HIV fusion to the target cell. HIV uses primarily CCR5.

11. Entry and Fusion inhibitors (2003-2007) 4 3 2 1 HIV requires two protein-protein interactions in order to bind and fuse to a target cell It first binds to CD4, then uses the chemokine receptor CCR5 (or CXCR4) to fuse.

12. Subsequent studies of “Exposed-Uninfected” individuals determined that a genetic defect in the CCR5 gene rendered them resistant to infection with HIV. Therefore, it was reasoned that mimicking the genetic defect with a drug may be a new strategy for blocking HIV replication. So began the hunt for entry inhibitors…

13. Entry inhibitors (2007) The class of anti-HIV drugs known as entry inhibitors are designed to block the interaction between the Envelope protein and the chemokine receptor. The majority of these drugs specifically block the interaction with CCR5. Only one drug is currently FDA-approved, Maraviroc.

14. Finally, the newest class of Anti-HIV drugs is Integrase inhibitors. (2007) These drugs prevent HIV from leaving a permanent DNA copy in the host cell genome. From: Integrase inhibitors to treat HIV/Aids Yves Pommier, Allison A. Johnson & Christophe Marchand Nature Reviews Drug Discovery 4, 236-248 (March 2005)

15. Limitations of Current HIV Therapy. • 1.) The Need for Combination Therapy • HIV mutates rapidly, and most drug resistance • mutations only require a single nucleotide change. Therefore, the use of multiple drugs at the same time decreases the chance the virus can mutate to escape all drugs at the same time. • 2.) Combination Treatment Won’t Cure HIV. • HIV drugs can only kill cells with active virus replication. Although combination therapy can maintain undetectable levels of the virus in blood indefintiely, if Rx is stopped the virus will resume replication as small numbers of resting infected cells become active again. Long-lived resting cells with integrated viral DNA serve as a source of recurrent virus whenever treatment is stopped. • In order to fully cure HIV infection all of these resting infected cells (known as the viral reservoir) must be eradicated.

16. The Berlin Patient N Engl J Med. 2009 Feb 12;360(7):692-8 Doctors in Germany replaced the bone marrow of an HIV+ man with the bone marrow of a CCR5 deficient (HIV-resistant) female donor. Now, more than 5 years later he still has no detectable HIV replication while not on therapy. Basically they killed the viral reservoir with toxic chemotherapy and replaced it with resistant cells.

17. Tim Brown is considered to be functionally cured of HIV infection, i.e. no detectable virus by any conventional means in the absence of treatment. Why can’t we do the same thing for all people infected with HIV? 1.) Transplant is high risk, 1 in 5 will die of a complication. 2.) There must be an appropriately matched bone marrow donor who is CCR5 32. 3.) The procedure requires high tech medical facilities and very long hospital stays.

18. Other approaches to curing HIV Gene therapy Aside from a full stem cell transplant, gene therapy may be used to deliver resistant cells to HIV-positive people. Or it can be used to deliver antibodies to protect cells from infection (more on this in the vaccine lecture). Mitsuyasu RT, et al. Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nat Med. 2009 Mar;15(3):285-92. Epub 2009 Feb 15. Nat Med. 2009 Aug;15(8):901-6

19. Other approaches to curing HIV “Shock and Kill” Nature. 2012 Jul 25;487(7408):482-5 Another approach to eradicating HIV is to “wake up” the resting cells with viral DNA (“shock”), then once they are making virus again they can be targeted to eradication (“kill”). Several drugs are currently being testing for their ability to stimulate the resting cells to make virus again.