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Partners in Science National Conference M.J.Murdock Charitable Trust San Diego, California January 12, 2007 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 We envision a world where people live free from the threat of infectious disease. [email protected] 1965 Science Fair International School of Bangkok 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 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 Million 25 years of Acquired Immunodeficiency Syndrome (AIDS) People living with HIV 50 45 40 35 30 Children orphaned by AIDS in sub-Saharan Africa 25 20 15 10 5 0 1980 1985 1990 1995 2000 2005 HIV-1 is variable worldwide Los Alamos Database http://hiv-web.lanl.gov/ Diversity on an INDIVIDUAL level Homogeneous new infection Replicates ~ 24hrs Produces 1010 new virions a day Average 1 mutation per replicated genome Rapidly develop a “quasispecies” Nancy L. Haigwood Seattle Biomedical Research Institute Nonhuman primate models for AIDS Macaca mulatta (rhesus) Macaca nemestrina (pigtailed) Macaca fascicularis (crab-eating) • 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 Nancy L. Haigwood Seattle Biomedical Research Institute 1% divergence Same virus, 11 macaques 89.6p consensus 89.6P 107 (0.3%) • • • • • 191 (0.3%) 098 099 (0.3%) 168 (0.4%) 098 (0.9%) 069 (1.2%) 071 (1.4%) 156 (1.8%) 246 (1.9%) 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? 108 (1.9%) 152 (1.9%) Nancy L. Haigwood Seattle Biomedical Research Institute (Blay, JV 2006) Infected cells producing HIV Images of HIV, Boehringer-Ingelheim Human Immunodeficiency Virus (HIV) http://mbim.web.arizona.edu/pics/hiv.gif Human Immunodeficiency Virus (HIV) http://mbim.web.arizona.edu/pics/hiv.gif Three levels of control in HIVexposed people Images of HIV, Boehringer-Ingelheim Activated cells with key receptors are vulnerable to HIV Monocyte with CD4 and CCR5 Nancy L. Haigwood Seattle Biomedical Research Institute Three types of “natural” resistance to infection 1. Genetic resistance to infection and activation 2. Innate immune responses 3. Acquired immunity Monocyte or macrophage with CD4 and CCR5 Nancy L. Haigwood Seattle Biomedical Research Institute Immunity to viral infection-clearance is key to health Illness Antibody CTL 0 Replication 7 14 28 35 Days post-infection Nancy L. Haigwood Seattle Biomedical Research Institute Acquired immunity: antibody-producing B cells Neutralizing (Env only) Non-neutralizing (all HIV proteins) Neutralizing antibodies are a subset of HIV-specific antibodies that block infection Nancy L. Haigwood Seattle Biomedical Research Institute Acquired immunity: cytotoxic T cells (CTL) CTL recognize viral proteins on the cell surface and destroy them Killer T-cell CTL recognize 9 specific amino acid sequences in all viral proteins--both variable and conserved antigens Nancy L. Haigwood Seattle Biomedical Research Institute 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 HIV binds to the cellular receptors Goal of drug treatment--stop the spread Nancy L. Haigwood Seattle Biomedical Research Institute Fusion inhibitors block infection Nancy L. Haigwood Seattle Biomedical Research Institute HIV fuses and releases its genome and enzymes Nancy L. Haigwood Seattle Biomedical Research Institute Inhibitors to RT and INT block infection and integration RT=reverse transcriptase IN=integrase Nancy L. Haigwood Seattle Biomedical Research Institute Cells are permanently infected with HIV Nancy L. Haigwood Seattle Biomedical Research Institute Infected cells produce more virus, not only in blood, but in tissues PR=protease Nancy L. Haigwood Seattle Biomedical Research Institute Protease inhibitors block HIV from becoming infectious Immature virions (noninfectious) Nancy L. Haigwood Seattle Biomedical Research Institute More cells are susceptible… Nancy L. Haigwood Seattle Biomedical Research Institute …and become infected Nancy L. Haigwood Seattle Biomedical Research Institute Now the reservoir is larger Nancy L. Haigwood Seattle Biomedical Research Institute More virus is made, and some are new variants gut blood brain Nancy L. Haigwood Seattle Biomedical Research Institute Viral reservoirs: drug- and CTLand antibody-resistant variants • • • • • Cells continue to produce virus in the reservoirs Without effective combination drugs, mutants arise New drugR strains can take over and be transferred No current drugs can eliminate the reservoir virus Unknown if immunity can be “enhanced” by therapeutic vaccination 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 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 Immunity to infection Illness Antibody CTL 0 Replication 7 14 28 35 Days post-infection Nancy L. Haigwood Seattle Biomedical Research Institute Goal of vaccination Live-attenuated vaccines are too risky! CTL Antibody Illness CTL Vaccine Antibody Infection on challenge or exposure Nancy L. Haigwood Seattle Biomedical Research Institute HIV Vaccine Approaches • Recombinant HIV proteins • DNA plasmids encoding HIV genes • Live-attenuated vector viruses expressing HIV proteins Ad • Chemically inactivated virions • Artificial virus-like particles HIV Vaccine Approaches http://www.nwabr.org/education/hiv.htm • Recombinant HIV proteins • T helper cells • DNA plasmids encoding HIV genes • Live-attenuated vector viruses expressing HIV proteins • Multiple HIV antigens • Binding Antibodies – Antibody-Dependent Cellular Cytotoxicity Ad • Chemically inactivated virions • Artificial virus-like particles • Neutralizing Antibodies – HIV Env – Block binding of virus to cell • Cytotoxic T Lymphocytes (CTL): – Target and kill virus-infected cells Nancy L. Haigwood Seattle Biomedical Research Institute 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 Variables in vaccine development 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 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 Design Dose Route Timing 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 Macaque Indian rhesus Chinese rhesus Crab-eating Pigtailed Courtesy of AS Fauci 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 Nancy L. Haigwood antibodies against multiple HIVs Seattle Biomedical Research Institute 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 HIV Envelope, master of disguise C1 V1V2 C2 V3 C3 V4 C4 V5 C5 5 conserved regions 5 variable regions Glycosylated (sugars on the outside of the protein) Nancy L. Haigwood Seattle Biomedical Research Institute HIV Env binds to CD4 to mediate entry http://www.prn.org/images/prn_nb_cntnt_images/vol7num1/eron_fig1a.gif • 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) Epitopes of broad NAbs from patients adapted from: Burton DR et al. PNAS 2005 gp41 4E10 2F5 gp120 IgG1b12 2G12 These NAbs “describe” specific regions of Env but they do not inform the mechanism of their development The blind men and the elephant: seeing the shape of HIV Envelope QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Photo of African elephants in Tanzania by William F. Sutton 1% divergence Same virus, 11 macaques 89.6p consensus 89.6P 107 (0.3%) 191 (0.3%) 098 099 (0.3%) 168 (0.4%) 098 (0.9%) 069 (1.2%) Are the envelope gene changes in each macaque similar or different? 071 (1.4%) 156 (1.8%) 246 (1.9%) 108 (1.9%) 152 (1.9%) Nancy L. Haigwood Seattle Biomedical Research Institute (Blay, JV 2006) PNGs altered in “hotspots” Prevalence of PNG Inoculum 1.0 0.8 0.6 0.4 0.2 0 V3 V1V2 V4 V5 Prevalence of PNG SHIV-89.6P 1.0 0.8 0.6 0.4 0.2 0 139C 141 V1V2 188 276 386 397 V3 V4 463 V5 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) Blay et al (2006) J Virol 80:999 PNG changes map proximal to CD4 binding site CD4bs CD4bs 139C 276 V5 V1V2 141 460 CD4bs 186 V1/V2 397 V4 V5 386 V3 V4 CD4bs view • Core crystal structure from Kwong and Wyatt, Nature, 1998 • Variable loops modeled by S. Gnanakaran, LANL • Image modified with Protein Explorer V3 Blay et al (2006) J Virol 80:999 PNG changes map proximal to CD4 binding site CD4bs CD4bs 139C 276 V5 V1V2 141 462 CD4bs 188 V1/V2 397 V4 V5 386 V3 V4 CD4bs view • Core crystal structure from Kwong and Wyatt, Nature, 1998 • Variable loops modeled by S. Gnanakaran, LANL • Image modified with Protein Explorer V3 Blay et al (2006) J Virol 80:999 Pseudovirus Production pEMC* 89.6p gp41 293T cells Variant gp120 HIV genome (with no env) Nancy L. Haigwood Seattle Biomedical Research Institute Neutralization assay • Tzm-bl cell line • Expresses CD4, CCR5, CXCR4 • HIV-1 LTR expression of -gal and luciferase • Infect with pseudovirus or patient HIV or SHIV HIV-1 LTR luc luciferase Relative Light Units Nancy L. Haigwood Seattle Biomedical Research Institute 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 The E2 Protein serves as a scaffold for a multienzyme complex PSBD Catalytic Domain (CD) Nancy L. Haigwood Seattle Biomedical Research Institute E2 antigen display system (E2DISP) Heterologous protein PSBD Catalytic Domain (CD) HP Adapted from Domingo et al., 2001. J Mol Biol 305:259. 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 “pure” particles Mix pure particles in varying ratios Denature in Guanidine HCl Renature “hybrid” particles Nancy L. Haigwood Seattle Biomedical Research Institute 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 Haigwood Lab, Summer 2004 Kristina Thorsen Sonali Mahalanabis Nicole Doria-Rose Nancy Haigwood QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Dina Lauman Wendy Blay Pushpa Jayaraman Not shown: Ruth Hotchkiss Jeanne Ting Chowning Bill Sutton Haigwood Lab, Summer 2006 Madhumita Mahalanabis Theresa Kasprzyk Dina Kovarik Pushpa Jayaraman, Ph.D. Bill Sutton Delphine Malherbe Ph.D. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Cherie Ng Mike Chester Wendy Blay, Ph.D. HIV and AIDS solutions 2007 • Behavior • Drug therapy • Vaccines • Education of the next generation • • • Partners in Science Light the path Shape the direction