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Unformatted text preview: Is there any hope for an antibody-based therapeutic approach against HIV given that people don’t normally make broadly neutralizing antibodies against HIV?
• HIV rapidly mutates so that antibodies are no longer effective. • HIV spike proteins are covered with carbohydrates, which are poorly or non-immunogenic. • Antibodies are too big to access some regions of the HIV spike. Burton, Dennis R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 14943-14948 HIV has evolved to thwart the immune system it has to confront
• Few, if any, HIV-infected individuals clear the virus. • We can, however, make neutralizing reagents (antibodies or antibody-like proteins) that prevent HIV infection. • What we cannot yet do is design an immunogen that will elicit broadly neutralizing reagents in a person (traditional vaccine approach). • Can we reprogram a human immune system so that it makes broadly neutralizing anti-HIV reagents? Name 4E10 Epitope Conserved (98%) linear WFXI sequence of gp41 Cterminal to the 2F5 epitope Conserved linear ELDKWA sequence of the membrane proximal ectodomain of gp41 Discontinous epitope overlapping the CD4 binding site of gp120 1 2 mannose cluster on the “silent” face of gp120 CD4-induced discontinuous epitope overlapping the coreceptor binding site CD4-induced discontinuous epitope near the coreceptor binding site Affinity (KD) 1 – 70 nM (n = 8) 0.5 – 5 nM (n = 8) 0.2 – 8 nM (n = 5) 2 nM (n = 1) 0.2 - 15 nM (n = 11) 0.1 – 100 nM (n = 3) Structure 2.2 Å Locations of antibody binding sites on schematic model of trimeric viral spike 2F5 2.0 Å b12 1.8 Å 1.8 Å 3.0 Å 2.5 Å 2G12 17b X5 (PD) 1.9 Å PD means “Phage display”, an in vitro method. Gene therapy -- could it be used to treat or prevent AIDS?
Adenovirus is not a retrovirus, so the inserted genes are not integrated into the host cell genome or inherited by the descendents of infected cell. Use recombinant DNA technology to insert a new gene into a virus, then use that to infect cells. Infected cells will transcribe the new gene and make the encoded protein.
http://en.wikipedia.org/wiki/Gene_therapy Gene therapy using retroviral vectors
• Advantage: Don’t have to re-administer the recombinant virus (as required if using recombinant adenoviruses) because genetic material is incorporated into the host cell genome, so all descendents of infected cell will inherit it. • Disadvantage: Retroviral integrase can insert the viral genetic material anywhere in the host genome.
• Insertion into the middle of a host cell gene will disrupt it. • Insertion upstream of a gene can disrupt its regulation. • Disruption of a gene regulating cell growth could lead to uncontrolled cell division (i.e., cancer). • Gene therapy trials in France to treat X-linked Severe Combined Immunodeficiency (X-SCID) were successful in curing the disease, but resulted in leukemia in several patients. Gene therapy may be safer using lentiviral vectors than onco-retroviral* vectors
leukemia virus (MLV)-based vector for ex vivo** infection of hematopoietic stem cells. • Lentiviral vectors (HIV,SIV) have different integration properties than the oncoretroviral vectors (MLV). • Infection with HIV is not known to cause integration-related cancers. • French trial treating X-SCID - 3 of 11 developed leukemia-like condition. Used murine from Hematti, P., et al. PLoS Biol 2: e234 (2004) * The prefix “onco” refers to oncology, which is the study of cancer. **ex vivo: Pertaining to a biological process or reaction taking place outside of a living cell or organism. Zinc finger endonuclease-mediated gene targeting in human cells Each “N” is a subunit of a dimeric endonuclease. Kandavelou et al. (2005) Nature Biotechnology 23: 686-687 • Zinc fingers that specifically target particular 3 basepair sequences were developed by phage display and other selection methods. • Because they are modular recognition units, they can be linked together in any order to create a reagent that specifically recognizes any desired DNA sequence. Gene Therapy
• Treat genetic diseases by correcting gene defect • Current gene therapy protocols involve gene addition to insert therapeutic genes randomly in genome using viral vectors -random insertions can activate oncogenes as seen in gene therapy trials to treat X-SCID. • Zinc finger endonuclease-mediated gene targeting uses cell’s homology-directed gene editing process rather than gene addition. • Dimerized enzyme introduces double-stranded break near site of mutation. Homologous recombination machinery repairs damage using wild-type gene that was introduced along with zinc finger endonuclease. Gene correction High, 2005, Nature 435; 577-579 Engineering Immunity against HIV
David Baltimore, PI, Bill and Melinda Gates Foundation
(Lentivirus) Hematopoietic stem cells Life-long supply of anti-HIV neutralizing antibodies Target HIV Lymphocytes Clicker question
Is it necessary to isolate autologous (a person’s own) hematopoietic stem cells in the Engineering Immunity approach?
1) Yes 2) No Smaller versions of antibodies against the CCR5 binding site on gp120 neutralize HIV more effectively than intact antibodies, but the immune system only produces intact antibodies
These are idealized data from an in vitro neutralization assay.
scFv (single-chain Fv) Intact IgG antibody Steric restrictions on antibody access to the coreceptor binding site on gp120
scFv and Fab versions of anti-CCR5 antibodies can fit in space between gp120 and target cell membrane. Intact IgG is too big. Explains why scFv and Fabs of anti-CCR5 antibodies neutralize virus better than IgG versions. People don’t normally make scFv and Fab versions of antibodies, but they could be programmed to do so in an Engineering Immunity approach. Burton et al. (2005) PNAS 102, 14943-8 Antibody design News feature, Nature (2007) volume 446, pp. 964-966 Anthony West, Josh Klein (former Bi1 TA) Engineering Immunity against HIV
David Baltimore, PI, Bill and Melinda Gates Foundation Is it practical to isolate hematopoietic stem cells from every person to be treated? Hematopoietic stem cells Life-long supply of anti-HIV neutralizing antibodies Target HIV Lymphocytes Baltimore/Yang/Wang Approach to Target Recombinant Lentiviruses to Hematopoietic Stem Cells (HSCs)
binding induces endocytosis
Receptor-mediated endocytosis brings recombinant lentivirus to an acid endosome. Anti-CD34 antibody targets lentivirus to HSCs. CD34
Recombinant lentivirus with membrane-bound anti-CD34 plus flu hemagglutinin, which has been mutated so that it no longer binds sialic acids. Low pH triggers hemagglutinin to fuse with endosomal membrane. endosomal compartment low pH
Viral capsid released into cytoplasm of cell. Potentially safer method than lentiviral or retroviral vectors: Use adeno-associated virus (AAV) to deliver genes • AAV infects humans and primates
– Doesn’t cause disease -- is a satellite virus
T = 1 icosahedral virus Crystal structure described in Xie et al., 2002, PNAS 99 10405-10 • ssDNA virus, stably integrates into host genome at specific site on chromosome 19 • Integrative capacity removed from AAV-based gene therapy vectors. Forms episomal (not in a chromosome) concatamers in host cell nucleus. – Concatamers retained in non-dividing cells
– Concatamers lost during cell division in non-dividing cells Johnson et al., 2009, Nature Medicine published online 17 May 2009; doi:10.1038/nm.1967 Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys • Immunized rhesus macaques with AAV vectors carrying neutralizing antibody constructs • Intramuscular injections
– achieved g/ml concentrations in the blood – single injection resulted in long-term (>1 year) expression of biologically-active antibodies • Four weeks after intramuscular injection, injected SIV • After six more weeks
– 6/6 control monkeys infected after challenge. 4/6 developed AIDS after ~one year – 6/9 AAV-injected monkeys were protected against infection, none developed AIDS after ~one year Extra credit opportunity
See course website for details • Submit an idea for how to prevent or cure HIV infection (due Friday 6/12/09). • If you wish to use an “Engineering Immunity” gene therapy approach (as discussed this lecture), follow the guidelines on the website instructions for describing your idea. • If you have a different sort of idea, follow the guidelines described for a Grand Challenges Exploration grant: go to http://www.gcgh.org/Explorations/Pages/ApplicationInstructions.aspx and click on “Create New Vaccines for Diarrhea, HIV, Malaria, Pneumonia and Tuberculosis”.
– The Bill and Melinda Gates Foundation is inviting anyone and everyone to submit short (maximum of two pages) proposals describing new ideas that are “off the beaten track” and “daring in premise”. – Applicants chosen for funding will receive $100,000 to carry out the research. Round 3 submissions was due May 28, 2009, but there will probably be another round of submissions in Fall 2009. – If anyone is interested in submitting his/her idea for Round 4, we will be happy to discuss it further with you and/or help you write the actual proposal. – If you are chosen for funding, we will find a laboratory at Caltech where you can conduct your research. If you are not chosen for funding, but are committed to trying your idea, we will help you find a way to test your idea in a lab at Caltech or elsewhere as a SURF project. Extra slides Antibody binding sites on model of trimeric viral spike Burton, Dennis R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 14943-14948 2G12 Fabs are domain swapped to form a rigid Fab2 unit.
Calarese et al., 2003, Science 300: 2065-71. 2G12 also forms domain-swapped dimers (two IgGs; four Fabs)
West et al., 2009, J. Virol. 83: 98-104 The dimeric form of 2G12 IgG is more effective in neutralizing HIV Dimer IC50: 0.94 ± 0.053 nM Monomer IC50: 43 ± 4.9 nM Dimer IC50: 0.43 ± 0.066 nM Monomer IC50: 30 ± 3.2 nM
Anthony West, Priyanthi Peiris, Joshua Klein 2G12 dimer neutralizes more effectively than 2G12 monomer
Env clone SC422661.8 TRO.11 PVO.4 QH0692.42 6535.3 TRJO4551.58 WITO4160.33 Average Monomer IC50 (μg/ml) 2.8 0.58 2.6 6.5 20 >100 6.1 6.2 Dimer IC50 (μg/ml) <0.05 <0.05 <0.05 <0.05 0.35 0.16 <0.05 0.08 Monomer IC50/ Dimer IC50 56 12 52 130 57 620 120 79 CAVD NAb Core, Beth Huey Tubman, Anthony West, Priyanthi Gnanapragasam ZFNs: Zinc finger chimeric endonucleases -- a way to make a site-specific double-stranded break at a chosen site
• Link zinc fingers to a nonspecific endonuclease (a dimer). • Use four fingers per endonuclease monomer, so effectively have a 24basepair recognition site -- long enough to specify unique site in mammalian genomes. • Zinc fingers that recognize specific triplets have been identified (most are in proprietary database of a biotech company called Sangamo BioSciences). • Use for gene therapy:
– Urnov et al. (2005) Nature 435: 646-651 (News and Views, pgs 577-579); Kandavelou et al. (2005) Nature Biotechnology 23: 686-687. Trimeric HIV envelope spike structures have been examined on viruses using cryoelectron tomography
The HIV envelope spike is a trimer of gp120/gp41* heterodimers. *gp (glycoprotein) 120 is 120 kDa; gp41 is 41 kDa. 3D reconstruction of SIV virions by cryo-electron tomography Zanetti, G., et al., PLoS Pathogens 2, e83 (2006) The resulting structure is low resolution, but shows that the trimer should be accessible to antibodies Zanetti, G., et al., PLoS Pathogens 2, e83 (2006) Zhu, P. , et al., Nature 441, 847 (2006) Clicker question: Companies have spent a lot of money trying to develop a soluble form of CD4 that could be injected into an HIV-positive patient. The reasoning behind this approach is that… (a) Soluble CD4 would compete with membrane bound CD4 to prevent gp120 from binding T cells. (b) Injected soluble CD4 is unlikely to trigger an immune reaction. (c) gp120 can’t mutate its CD4 binding site without reducing its ability to infect T cells. (d) All of the above. (e) None of the above. gp120 CD4 CCR5 Clicker question: Companies have spent a lot of money trying to develop a soluble form of CD4 that could be injected into an HIV-positive patient. The reasoning behind this approach is that… (a) Soluble CD4 would compete with membrane bound CD4 to prevent gp120 from binding T cells. (b) Injected soluble CD4 is unlikely to trigger an immune reaction against it. (c) gp120 can’t mutate its CD4 binding site without reducing its ability to infect T cells. (d) All of the above. (e) None of the above. soluble CD4 Ping Zhu, Ken Roux, Univ. Florida Mutant SIV ~75 trimers HIV 7-14 trimers 100 nm ...
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