Preventing the Spread of HIV by Vector-Encoded Antibody
Preventing the Spread of HIV by Vector-Encoded Antibody Cancer Research in the 21st Century David Baltimore UCL Cancer Institute Dedication K/RITH Opening 18Oct, Sept2012 07 11 David Baltimore A 30-Year-Old Challenge: Making A Vaccine Against HIV Because the virus takes years, if ever, to elicit from a patient’s body antibodies that might stop the infection– and then it is too late— and T cells do not sufficiently control the virus, making a vaccine has been a daunting task. The Big Advance • Even though they do not protect the person from whom they were isolated, broadly neutralizing antiHIV human monoclonal Abs have been found, isolated and studied in detail – Some are very broad and quite potent and more coming weekly • So the issue becomes, how do we build on this demonstration that the human repertoire can make a potentially prophylactic antibody? • Remember the antibodies: Broadly Neutralizing Antibodies Against Env PG9, PG16, PGT121, PGT128 (Variable Loops 1,2,3) b12, VRC01, NIH45-46 (CD4 Binding Site) 2G12 (Glycanbinding) 2F5, 4E10 (Membrane Proximal) Antibodies from the VRC (Nabel), IAVI (Burton), Rockefeller (Nussenzweig) How can we use these Abs to provide anti-HIV prophylaxis? • We could try to elicit them • We could make them in large amounts and inject them • We could use viral vectors expressing the appropriate genes to program the body to make the antibodies • We call this VIP (Vectored ImmunoProphylaxis) AAV Mediated Delivery of Neutralizing Antibodies as Prophylaxis Against HIV: Vectored ImmunoProphylaxis (VIP) Alex Balazs Engineering Ab Production using AAV8 o Positive Characteristics of Adeno-Associated Virus o Non-pathogenic in humans o Non-integrating DNA virus 20nm giving long-lived expression following a single administration o Excellent expression ability in vivo in animals and suggestive data in humans o Grown to high titer by transient transfection o Negative characteristic: very small (5 kB) HJ Nam et al. Our AAV Transfer Vector 2-5x Luciferase CASI SV40pA WPRE 10x oPromoter oPolyadenylation Signal oAdditional Post-transcriptional regulatory elements (WPRE) ITR CASI Prom. IgG1 HC 2A IgG1 LC AAV IgG Expression Vector - 4.5 kb WPRE SV40pA ITR AAV2/8-Mediated Luciferase Expression Intramuscular Injection Intravenous Injection 107 108 109 Photons/sec/cm2 1010 AAV2/8 Mediates Long-Term Gene Expression in Immunodefficient (Rag2/γc-/-) Mice 107 108 109 Photons/sec/cm2 1010 NOD/SCID/γ-/- HuPBMC Model as a Model of CD4 Depletion During HIV Infection Transfer Human PBMCs 2 Wks Challenge with HIV Sample Weekly Experimental Design to Test Antibodies In Vivo • Administer antibody expression vectors or control • Humanize immunodeficient mice by engrafting with PBMCs • Challenge with HIV • Quantify CD4/CD8 ratios over time Inject AAV D-35 Inject HuPBMC HIV Challenge IV or IP -21 0 Expand PBMC 13 Days D-33 -21 Blood Sampling 7 14 21 28 35 42 49 56 b12 Expressed from CASI Driven AAV Vector Protects Animals from CD4 Loss Following Intraperitoneal HIV Challenge Histology of NL4-3 Challenged Humanized Mice Luciferase B12 o No evidence of p24+ cells in spleens of mice treated with B12 The Recently Identified VRC01 Antibody Neutralizes a Broad Array of HIV Strains Engrafted Human CD4 Cells are Protected by VRC01 N=8-12 Determination of the Minimum Protective VRC01 Dose In Vivo • 8.3 µg/mL of VRC01 was sufficient to protect • 1.6 µg/mL of VRC01 was not sufficient to protect • The intravenous challenge dose represents approximately 1x108 physical particles of CXCR4tropic NL4-3 Open Questions • Will VIP work against R5 virus? • Will VIP work against founder strains? • Will VIP work against a mucosal challenge? VRC01 Protects Against Infection by an R5 Transmitted Founder HIV Strain (REJO.c) REJO.c NL43 Luciferase b12 VRC01 To examine protection against mucosal infection we went to a different model, the BLT mouse [Mouse with a fetal human liver/ thymus graft populated with CD34+ hematopoietic stem cells] Comparison of NSG-HuPBMC and BLT Mouse Models NSG-HuPBMC BLT ~ 8 Weeks ~ 24 Weeks Engrafted lineages T-Cells All Lineages Host tissue engraftment Limited Extensive Non-functional Some function Thousands 20-30 Maximum Difficulty of production Low High Cost per animal Low High Duration of engraftment Immune Function Potential group size from single donor Can VIP Protect a Model of Human-to-Human Transmission? • Create BLT mice (Dong Song An, UCLA) • Administer vector intramuscularly • Challenge intravaginally with HIV repetitively • Sample repetitively Surgical Implantation of Bone-LiverThymus to produce BLT Animal Inject AAV -70 -28 Repetitive “Low-Dose” Intravaginal Challenge with 50ng JR-CSF Harvest Tissue 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98105 Blood Sampling for 15 Weeks VRC01 Antibody is Detected in Vaginal Secretions Serum Detection Limit = 70ng/mL Vaginal Wash Detection Limit = 1.3ng/mL Can this level of antibody protect mice from mucosal challenge? VRC01 Protects Against Loss of CD4 Cells in BLT Mice Luciferase VRC01 VRC01 Protects Against Mucosal HIV Transmission Splenic Lymphocytes Vaginal Lamina Propria Lymphocytes Plasma Viral Load Detection Limit = 200 Copies/mL VRC01 Reduces Risk of HIV Transmission Through Mucosal Route Detection Limit = 1500/mL VRC01 Reduces Risk of HIV Transmission Through Mucosal Route Can VIP Protect Against a Transmitted Founder Strain? • Create BLT mice • Administer vector expressing VRC07G54W antibody • Challenge intravaginally with a Transmitted Founder Strain of HIV (REJO.c) Surgical Implantation of Bone-LiverThymus to produce BLT Inject AAV Animal -70 -28 Repetitive “Low-Dose” Intravaginal Challenge with REJO.c 0 Harvest Tissue 7 14 21 28 35 42 49 56 63 70 77 84 81 88 95 102109116 123130 Blood Sampling for 20 Weeks VRC07G54W Antibody is Detected in Vaginal Secretions Serum Detection Limit = 70ng/mL Vaginal Wash Detection Limit = 1.3ng/mL VRC07G54W Protects Against Loss of CD4 Cells in BLT Mice Luciferase VRC07G54W VRC01 Protects Against Mucosal HIV Transmission Gut IntraSplenic Epithelial Lymphocytes Lymphocytes Gut Lamina Propria Lymphocytes Vaginal Lamina Propria Lymphocytes VIP-Treated Mice Show No Detectable HIV Despite Extensive Mucosal Challenge Plasma Viral Load Detection Limit = 200 Copies /mL Conclusions • Vectored Immunoprophylaxis is capable of protecting animals from X4 and R5 strains, including a transmitted molecular founder strain. • This approach protects humanized mice from repeated muscosal challenge. Transitioning to Clinical Development • Today we know enough to be confident that if humans act like mice we can protect humans against HIV infection • To test human responses we have teamed up with the Gary Nabel and his team at the VRC at NIH to run clinical trials • First need to ask if humans make prophylactic levels of Ab • Need to carefully investigate safety (therapeutic antibodies have proved quite safe) VIP • VIP was developed to prevent HIV infection and does this effectively in humanized mice • VIP could have wider applicability, for instance in preventing the spread of influenza virus • It is relatively cheap and quite stable making it appropriate for use in the developing world as well as stock-piling against natural or bioterrorist-mediated viral diseases People Who Did This Work • Alex Balazs with the help of Joyce Chen, Christin Hoag and Stella Ouyang • Lili Yang, project manager of Engineering Immunity Program
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