2. Why Vaccines and Therapies for HIV are So Challenging: New Strategies to Outwit the Virus Nancy L. Haigwood, Ph.D. Seattle Biomedical Research Institute University of Washington Partners in Science National Conference M.J.Murdock Charitable Trust San Diego, California January 12, 2007 We envision a world where people live free from the threat of infectious disease. Nancy.haigwood@sbri.org

3. 1965 Science Fair International School of Bangkok

4. The epidemic, the virus, the genome, molecular structures Mysteries of HIV, master of escape Progress in drug therapy Progress in vaccine development Role for science teaching and inspiration at all levels

5. HIV and AIDS Human Immunodeficiency Virus (HIV) is the virus AIDS = Acquired Immune Deficiency Syndrome –Consequence of HIV infection –Immune system unable to fight other diseases Death due to opportunistic infections –Those that don’t usually cause illness Illness and death after ~10-20 years –Cannot eradicate the virus once infected –100% mortality

6. 25 years of Acquired Immunodeficiency Syndrome (AIDS) First cases of unusual immune deficiency identified among gay men in USA 40 30 20 10 0 50 35 25 15 5 45 Million 19801985199019952000 2005 AIDS defined for the first time HIV identified as the cause of AIDS In Africa, a heterosexual AIDS epidemic is revealed The first HIV antibody test becomes available zidovudine, or AZT approved for use in USA People living with HIV Children orphaned by AIDS in sub-Saharan Africa HAART launched

7. HIV-1 is variable worldwide Los Alamos Database http://hiv-web.lanl.gov/

8. Diversity on an INDIVIDUAL level Average 1 mutation per replicated genome Homogeneous new infection Replicates ~ 24hrs Produces 10 10 new virions a day Rapidly develop a “quasispecies” Nancy L. Haigwood Seattle Biomedical Research Institute

9. Nonhuman primate models for AIDS HIV-1: only replicates in chimpanzees--disease in 10 years SIV: simian immunodeficiency virus; transferred from African to Asian macaques in captivity and caused disease like AIDS SHIV: chimera that has the HIV Envelope and the backbone of SIV; these viruses cause disease after passage in macaques Macaca mulatta (rhesus) Macaca nemestrina (pigtailed) Macaca fascicularis (crab-eating) Nancy L. Haigwood Seattle Biomedical Research Institute

10. 098 (0.9%) 069 (1.2%) 071 (1.4%) 156 (1.8%) 246 (1.9%) 108 (1.9%) 152 (1.9%) 1% divergence 89.6p consensus 191 (0.3%) 099 (0.3%) 107 (0.3%) 89.6P 168 (0.4%) 098 Same virus, 11 macaques Envelope DNA sequences Approx 10 clones per macaque 32 weeks post-infection Maximum likelihood tree Average % divergence in parentheses Each monkey has a different virus collection termed “quasispecies” Some animals have more virus and more divergence--why? (Blay, JV 2006) Nancy L. Haigwood Seattle Biomedical Research Institute

11. Infected cells producing HIV Images of HIV, Boehringer-Ingelheim

12. Human Immunodeficiency Virus (HIV) http://mbim.web.arizona.edu/pics/hiv.gif

13. Human Immunodeficiency Virus (HIV) http://mbim.web.arizona.edu/pics/hiv.gif

14. Three levels of control in HIV- exposed people Images of HIV, Boehringer-Ingelheim

15. Activated cells with key receptors are vulnerable to HIV Nancy L. Haigwood Seattle Biomedical Research Institute Monocyte with CD4 and CCR5

16. Three types of “natural” resistance to infection 1.Genetic resistance to infection and activation 2.Innate immune responses 3.Acquired immunity Nancy L. Haigwood Seattle Biomedical Research Institute Monocyte or macrophage with CD4 and CCR5

17. Days post-infection Antibody 71428350 Illness CTL Replication Immunity to viral infection-- clearance is key to health Nancy L. Haigwood Seattle Biomedical Research Institute

18. Acquired immunity: antibody-producing B cells Non-neutralizing (all HIV proteins) Neutralizing (Env only) Neutralizing antibodies are a subset of HIV-specific antibodies that block infection Nancy L. Haigwood Seattle Biomedical Research Institute

19. Killer T-cell Acquired immunity: cytotoxic T cells (CTL) CTL recognize 9 specific amino acid sequences in all viral proteins--both variable and conserved antigens Nancy L. Haigwood Seattle Biomedical Research Institute CTL recognize viral proteins on the cell surface and destroy them

20. Immunity in HIV infection Local innate responses may control infection in some cases (e.g. sex workers in Africa) Robust cellular immunity associated with long-term control and low virus loads Robust neutralizing antibodies can block infection if there before exposure and also help control Infection is not cleared by these mechanisms and the virus persists in the host DNA in blood and other reservoirs Loss of gut lymphocytes an early event that is critical to survival and immunity Nancy L. Haigwood Seattle Biomedical Research Institute

21. HIV binds to the cellular receptors Goal of drug treatment--stop the spread Nancy L. Haigwood Seattle Biomedical Research Institute

22. Fusion inhibitors block infection Nancy L. Haigwood Seattle Biomedical Research Institute

23. HIV fuses and releases its genome and enzymes Nancy L. Haigwood Seattle Biomedical Research Institute

24. Inhibitors to RT and INT block infection and integration RT=reverse transcriptase IN=integrase Nancy L. Haigwood Seattle Biomedical Research Institute

25. Cells are permanently infected with HIV Nancy L. Haigwood Seattle Biomedical Research Institute

26. Infected cells produce more virus, not only in blood, but in tissues Nancy L. Haigwood Seattle Biomedical Research Institute PR=protease

27. Protease inhibitors block HIV from becoming infectious Immature virions (noninfectious) Nancy L. Haigwood Seattle Biomedical Research Institute

28. More cells are susceptible… Nancy L. Haigwood Seattle Biomedical Research Institute

29. …and become infected Nancy L. Haigwood Seattle Biomedical Research Institute

30. Now the reservoir is larger Nancy L. Haigwood Seattle Biomedical Research Institute

31. More virus is made, and some are new variants Nancy L. Haigwood Seattle Biomedical Research Institute blood gut brain

32. Viral reservoirs: drug- and CTL- and antibody-resistant variants Cells continue to produce virus in the reservoirs Without effective combination drugs, mutants arise New drug R strains can take over and be transferred No current drugs can eliminate the reservoir virus Unknown if immunity can be “enhanced” by therapeutic vaccination

33. Progress in drug discovery Better understanding of viral regulation More activity in drug discovery (new targets) Additional animal models for other targets Experience in the developing world Drugs more widely available (political pressure) Better application of drugs- (time, dose, patient) –Monitor for drug-resistance and adjust “cocktail” –Reduce Mother-to-Child-Transmission –Lengthen lives by earlier drug application –Reduce transmission Nancy L. Haigwood Seattle Biomedical Research Institute

34. HIV Vaccine Goals Goal is CONTROL virus load: –Would work like anti-HIV drug “cocktail” –Could slow the time to onset of AIDS –Lengthen lives –Reduce transmission to others

35. Days post-infection Antibody 71428350 Illness CTL Replication Immunity to infection Nancy L. Haigwood Seattle Biomedical Research Institute

36. VaccineInfection on challenge or exposure CTL Antibody CTL Antibody Illness Goal of vaccination Nancy L. Haigwood Seattle Biomedical Research Institute Live-attenuated vaccines are too risky!

37. HIV Vaccine Approaches Recombinant HIV proteins DNA plasmids encoding HIV genes Live-attenuated vector viruses expressing HIV proteins Chemically inactivated virions Artificial virus-like particles Ad

38. Recombinant HIV proteins DNA plasmids encoding HIV genes Live-attenuated vector viruses expressing HIV proteins Chemically inactivated virions Artificial virus-like particles Ad Multiple HIV antigens T helper cells Binding Antibodies –Antibody-Dependent Cellular Cytotoxicity Neutralizing Antibodies –HIV Env –Block binding of virus to cell Cytotoxic T Lymphocytes (CTL): –Target and kill virus-infected cells HIV Vaccine Approaches http://www.nwabr.org/education/hiv.htm Nancy L. Haigwood Seattle Biomedical Research Institute

39. HIV Vaccine Progress Vaccines can reduce virus loads in nonhuman primate challenge models  Work like anti-HIV drug “cocktails”  Slow the time to onset of symptoms  Eventually all succumb to AIDS  Can be superinfected (also in humans)  Vaccines that “work” best elicit innate, cellular, and humoral immunity (neutralizing antibodies) Nancy L. Haigwood Seattle Biomedical Research Institute

40. Vector Vaccinia MVA Fowlpox Canarypox Adenovirus VEE Semiliki Forest Virus Adeno-associated virus OPV Herpes simplex virus Rabies virus VSV Measles Moloney Leukemia virus Hepatitis B virus Listeria monocytogenes BCG Salmonella Gene Env Gag Rev Tat Nef Pol Vpu Vif Vpr Virus SIVmac.239/251 SIV E660 SIV mne E11S SIV pbj14 SIVDB670 SHIV 89.6P SHIV SF 162P SHIV DH12R SHIV E-P4 SHIV Grt1FX SHIV KU2 SHIV.C2.\1 Vaccine Mode Whole, killed Attenuated DNA Recomb. proteins Peptides Lipopeptides Mimetopes Pseudovirions Virus-like particles Vector Adjuvant** Alum Cytokines Pulsed DC Co-stimulatory QS-21 CpG oligos PROPRIETARY Macaque Indian rhesus Chinese rhesus Crab-eating Pigtailed Design Dose Route Timing Variables in vaccine development

41. Courtesy of AS Fauci

42. HIV Vaccine Challenges Vaccines elicit low level immunity humans  Similar to macaque studies in magnitude, duration, types of immunity  Combination vaccines better than single modality  No protection in first vaccine trial (Env protein only) NO CHALLENGES  Excitement over adenovirus vector delivery  Still seeking ways to generate neutralizing antibodies against multiple HIVs Nancy L. Haigwood Seattle Biomedical Research Institute

43. HIV Envelope, master of disguise Envelope spike = 3 functional Envelope proteins (gp160) gp160 = gp120 surface unit, gp41 transmembrane unit Nancy L. Haigwood Seattle Biomedical Research Institute

44. 5 conserved regions 5 variable regions Glycosylated (sugars on the outside of the protein) V1V2 V3 V4 V5 C2 C1 C3 C4 C5 HIV Envelope, master of disguise Nancy L. Haigwood Seattle Biomedical Research Institute

45. http://www.prn.org/images/prn_nb_cntnt_images/vol7num1/eron_fig1a.gif HIV Env binds to CD4 to mediate entry HIV Env must bind to CD4 to gain entry into the host cell This is theoretically a weak point for the virus, which could also be targeted by neutralizing antibodies (NAbs)

46. gp41 gp120 Epitopes of broad NAbs from patients 4E10 2F5 IgG1b12 2G12 adapted from: Burton DR et al. PNAS 2005 These NAbs “describe” specific regions of Env but they do not inform the mechanism of their development

47. The blind men and the elephant: seeing the shape of HIV Envelope Photo of African elephants in Tanzania by William F. Sutton

48. 098 (0.9%) 069 (1.2%) 071 (1.4%) 156 (1.8%) 246 (1.9%) 108 (1.9%) 152 (1.9%) 1% divergence 89.6p consensus 191 (0.3%) 099 (0.3%) 107 (0.3%) 89.6P 168 (0.4%) 098 Same virus, 11 macaques Are the envelope gene changes in each macaque similar or different? (Blay, JV 2006) Nancy L. Haigwood Seattle Biomedical Research Institute

49. N-glycosite (LANL) analysis of 89.6P 102 SHIV-89.6P gp120 clones 19/23 PNGs conserved in >90% of sequences, found on silent face of gp120 in crystal structure 6 PNGs altered in V1/V2, C2, V, V5 All are under selective pressure (dN/dS >1) 0 0.2 0.4 0.6 0.8 1.0 Inoculum Prevalence of PNG V1V2 V3V4 V5 0 0.2 0.4 0.6 0.8 1.0 SHIV-89.6P 141 139C 188 276386397 463 V1V2 V3V4 V5 Prevalence of PNG PNGs altered in “hotspots” Blay et al (2006) J Virol 80:999

50. V3 V1V2 V4 V5 CD4bs 397 386 276139C 141 460 186 V3 V1/V2 V4 V5 CD4bs CD4bs view Core crystal structure from Kwong and Wyatt, Nature, 1998 Variable loops modeled by S. Gnanakaran, LANL Image modified with Protein Explorer Blay et al (2006) J Virol 80:999 PNG changes map proximal to CD4 binding site

51. V1V2 V4 V5 CD4bs 397 386 276139C 141 462 188 V3 V1/V2 V4 V5 CD4bs CD4bs view Core crystal structure from Kwong and Wyatt, Nature, 1998 Variable loops modeled by S. Gnanakaran, LANL Image modified with Protein Explorer Blay et al (2006) J Virol 80:999 PNG changes map proximal to CD4 binding site

52. Pseudovirus Production HIV genome (with no env) 293T cells pEMC* 89.6p gp41 Variant gp120 Nancy L. Haigwood Seattle Biomedical Research Institute

53. Neutralization assay HIV-1 LTR luc luciferase Relative Light Units Tzm-bl cell line Expresses CD4, CCR5, CXCR4 HIV-1 LTR expression of  -gal and luciferase Infect with pseudovirus or patient HIV or SHIV Nancy L. Haigwood Seattle Biomedical Research Institute

54. Conclusions from Envelope studies: HIV Envelope is plastic and utilizes many methods to evade neutralizing antibodies (sugars, charge change, deletions) There is a LIMIT to where and how much change can be tolerated (Achilles’ heel) Expose conserved regions Nancy L. Haigwood Seattle Biomedical Research Institute

55. The E2 Protein serves as a scaffold for a multienzyme complex PSBD Catalytic Domain (CD) Nancy L. Haigwood Seattle Biomedical Research Institute

56. E2 antigen display system (E2DISP) PSBD Catalytic Domain (CD) Heterologous protein HP Adapted from Domingo et al., 2001. J Mol Biol 305:259.

57. E2DISP-HIV Particles: pure and hybrid Express E2DISP-HIV molecules in E. coli BL21 cultures Purification of 60mers: ammonium sulfate fractionation, ion exchange chromatography and gel filtration Mix pure particles in varying ratios Denature in Guanidine HCl Renature “pure” particles “hybrid” particles Nancy L. Haigwood Seattle Biomedical Research Institute

58. E2DISP Antigen Display System Advantages –Ability to present up to 60 different heterologous proteins on surface of artificial Virus Like Particles –Present epitopes in unique conformations (conserved Env!) –Elicit antibodies, T cell help and CTL –Obtain up to 3 mg protein per 1 L culture –Antibodies are long-lived and high titer Limitations –Prokaryotic system: No glycosylation –Each construct requires optimization of purification –Difficulties characterizing exact particle composition –Will get responses to E2 also; best used in combination

59. Haigwood Lab, Summer 2004 Nicole Doria-Rose Kristina Thorsen Nancy Haigwood Bill Sutton Sonali Mahalanabis Pushpa Jayaraman Wendy Blay Dina Lauman Not shown: Ruth Hotchkiss Jeanne Ting Chowning

60. Pushpa Jayaraman, Ph.D. Cherie Ng Bill Sutton Wendy Blay, Ph.D. Mike Chester Delphine Malherbe Ph.D. Dina Kovarik Theresa Kasprzyk Madhumita Mahalanabis Haigwood Lab, Summer 2006

61. HIV and AIDS solutions 2007 Behavior Drug therapy Vaccines Education of the next generation Partners in Science Light the path Shape the direction