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723. Ad5-Ad48 Hexon Hypervariable Region Substitutions Affect Viral Biodistribution and Inflammatory Responses In Vivo
ADENOVIRUS AND OTHER DNA VIRUS VECTORS III 723. Ad5-Ad48 Hexon Hypervariable Region Substitutions Affect Viral Biodistribution and Inammatory Responses In Vivo
Lynda Coughlan, Angela Bradshaw, Laura Denby, Stuart A. Nicklin,1 Jerome Custers,2 Nico van Rooijen,3 Dan H. Barouch,4 Andrew H. Baker.1 1 BHF GCRC, University of Glasgow, Glasgow, United Kingdom; 2 Crucell, Leiden, Netherlands; 3Department of Molecular Cell Biology, Vrije Universiteit Medical Centre, Amsterdam, Netherlands; 4Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston. 1
1
1
Human adenovirus type-5 (Ad5) is currently in development for various clinical gene therapy and vaccine applications. However, a number of challenges remain to be overcome for its optimisation. A high afnity interaction between Ad5 hexon and coagulation factor FX mediates hepatocyte transduction. Additionally, the high seroprevalence of Ad5 neutralising antibodies in humans may limit clinical efcacy. Ad48, a subgroup D adenovirus with low seroprevalence in humans, does not interact with FX. Substitution of the seven hypervariable regions (HVRs) within the Ad5 hexon for those of Ad48 produced a hexon chimeric vector, Ad5HVR48(1-7), which failed to bind FX by SPR. In this study, we compared the in vivo pharmacokinetics of Ad5 and Ad5HVR48(1-7) in macrophagedepleted and untreated control mice. Mice were injected intravenously with 3x1010vp of each virus, sacriced 1h and 48h post-injection and various organs harvested for analysis. Inammatory cytokine levels in plasma were determined 6h post-injection. There were no differences in the number of Ad5 and Ad5HVR48(1-7) genomes sequestered in the liver 1h post-infection when comparing control and macrophage-depleted animals. By 48h, the number of Ad5 and Ad5HVR48(1-7) genomes in the livers of control animals did not differ. However, signicantly higher levels of Ad5 genomes were detected in the livers of macrophage-depleted animals (>8-fold higher, p<0.01). Conversely, macrophage depletion signicantly attenuated the number of Ad5HVR48(1-7) genomes detected in the liver when compared to untreated animals (∼5-fold lower, p<0.001). In the spleens of control animals 1h post-injection, Ad5HVR48(1-7) genome levels were ∼3-fold higher than Ad5 (p=0.01). Splenic sequestration of Ad5 and Ad5HVR48(1-7) virions were unaffected by macrophage depletion. At 48h, Ad5HVR(1-7) genome levels in the spleen were ∼10-fold higher than Ad5 (p<0.05). In macrophage-depleted animals, Ad5 levels in the spleen were increased ∼12-fold compared to control (p<0.05). Macrophage depletion did not affect accumulation of Ad5HVR48(1-7) in the spleen. Additionally, cytokines including IL-6, IL-12, TNF-α, IFN-γ, were elevated in control animals injected with Ad5HVR48(1-7), but not Ad5. IL-6 and IL-12 levels induced by Ad5HVR48(1-7) were reduced upon macrophage depletion. These data suggest that large hexon modications can lead to an unpredictable biological outcome. This highlights the importance of designing vectors with smaller, dened mutations to fully optimise the biodistribution and toxicity prole of Ad-based vectors.
724. Evaluation of a Porcine Adenovirus 3 Vector as a Post-Exposure Vaccine Candidate
Ami Patel,1,2 Suresk K. Tikoo,3 Gary P. Kobinger.1,2 1 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada; 2Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3Vaccine and Infectious Disease Organization/University of Saskatchewan, Saskatoon, SK, Canada. Experimental adenovirus (Ad) vectors are promising vaccine platforms capable of stimulating robust humoral and cellular immune responses. In particular, human adenovirus 5 (AdHu5) based vaccines have afforded successful protection against emerging pathogens including inuenza, Ebola, and SARS. The generation of rapid protective immune responses against emerging zoonotic pathogens may be advantageous for vaccination and post-exposure treatment of at-risk populations including healthcare and support workers. Unfortunately, a large percentage of the human population has naturally-acquired pre-existing immunity against AdHu5 which can reduce vaccine efcacy and potential for vector readministration. To address this issue novel vector platforms are being developed using rare human and other mammalian adenovirus serotypes with low seroprevalence in humans. The current study evaluates protection and immune responses following vaccination against avian inuenza H5N1 with porcine adenovirus 3 (PAV3) as an alternative vehicle with low seroprevalence in the human population. An optimized expression cassette containing the A/Hanoi/30408/2005 hemagglutinin (HA) gene was inserted into a replication incompetent PAV3 vector by homologous recombination (PAV3-HA). Protection was monitored in BALB/c mice following lethal challenge with homologous H5N1 virus and an AdHu5-HA vaccine was also developed in parallel for comparison. PAV3-HA and AdHu5-HA shared similar protective efcacy against lethal challenge 28-days post-vaccination. Additionally, hemagglutination inhibition (HI) and neutralizing antibody (NAB) responses were comparable for both vaccines, although NAB titers were signicantly higher for PAV3HA at early days post-vaccination. Protection against inuenza is strongly mediated by the humoral response however a good cellular response can accelerate viral clearance. T-cell responses detected by the production of interferon-gamma were signicantly higher for the PAV3-HA vaccine early post-immunization when compared to Adhu5-HA. The generation of fast T and B-cell immune responses suggested that the PAV3 vector may be a good candidate for inducing rapid protective immunity and therefore successful post-exposure vaccination. To address this possibility, mice were immunized with the PAV3-HA vaccine at 5, 8 or 10 days before being submitted to a lethal challenge with H5N1 virus or 1 hour after challenge. Survival and virus titer in the lungs were compared with the AdHu5-HA vaccine. Interestingly, lung homogenate from PAV3-HA vaccinated animals had lower average virus titers on day 3 following challenge. Overall, PAV3-based vectors may offer a promising alternative to traditional AdHu5 vectors for usual prophylaxis vaccination in addition to the rapid induction of protective immune responses after exposure to infectious pathogens.
725. The Knob Domain Replacement Strategy Is Broadly Applicable to a Wide Variety of Adenovirus Fiber Proteins Galina Mikheeva, Natalya Belousova, Victor Krasnykh. Experimental Diagnostic Imaging, The University of Texas M.D. Anderson Cancer Center, Houston, TX.
The inability of adenovirus (Ad) vectors to deliver genes to cells in a target-specic manner remains one of the major limitations of these vectors. Previous successes in Ad targeting were largely limited to vectors derived from human Ad serotype 5 (Ad5). The practical S282
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Lynda Coughlan, Angela Bradshaw, Laura Denby, Stuart A. Nicklin,1 Jerome Custers,2 Nico van Rooijen,3 Dan H. Barouch,4 Andrew H. Baker.1 1 BHF GCRC, University of Glasgow, Glasgow, United Kingdom; 2 Crucell, Leiden, Netherlands; 3Department of Molecular Cell Biology, Vrije Universiteit Medical Centre, Amsterdam, Netherlands; 4Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston. 1
1
1
Human adenovirus type-5 (Ad5) is currently in development for various clinical gene therapy and vaccine applications. However, a number of challenges remain to be overcome for its optimisation. A high afnity interaction between Ad5 hexon and coagulation factor FX mediates hepatocyte transduction. Additionally, the high seroprevalence of Ad5 neutralising antibodies in humans may limit clinical efcacy. Ad48, a subgroup D adenovirus with low seroprevalence in humans, does not interact with FX. Substitution of the seven hypervariable regions (HVRs) within the Ad5 hexon for those of Ad48 produced a hexon chimeric vector, Ad5HVR48(1-7), which failed to bind FX by SPR. In this study, we compared the in vivo pharmacokinetics of Ad5 and Ad5HVR48(1-7) in macrophagedepleted and untreated control mice. Mice were injected intravenously with 3x1010vp of each virus, sacriced 1h and 48h post-injection and various organs harvested for analysis. Inammatory cytokine levels in plasma were determined 6h post-injection. There were no differences in the number of Ad5 and Ad5HVR48(1-7) genomes sequestered in the liver 1h post-infection when comparing control and macrophage-depleted animals. By 48h, the number of Ad5 and Ad5HVR48(1-7) genomes in the livers of control animals did not differ. However, signicantly higher levels of Ad5 genomes were detected in the livers of macrophage-depleted animals (>8-fold higher, p<0.01). Conversely, macrophage depletion signicantly attenuated the number of Ad5HVR48(1-7) genomes detected in the liver when compared to untreated animals (∼5-fold lower, p<0.001). In the spleens of control animals 1h post-injection, Ad5HVR48(1-7) genome levels were ∼3-fold higher than Ad5 (p=0.01). Splenic sequestration of Ad5 and Ad5HVR48(1-7) virions were unaffected by macrophage depletion. At 48h, Ad5HVR(1-7) genome levels in the spleen were ∼10-fold higher than Ad5 (p<0.05). In macrophage-depleted animals, Ad5 levels in the spleen were increased ∼12-fold compared to control (p<0.05). Macrophage depletion did not affect accumulation of Ad5HVR48(1-7) in the spleen. Additionally, cytokines including IL-6, IL-12, TNF-α, IFN-γ, were elevated in control animals injected with Ad5HVR48(1-7), but not Ad5. IL-6 and IL-12 levels induced by Ad5HVR48(1-7) were reduced upon macrophage depletion. These data suggest that large hexon modications can lead to an unpredictable biological outcome. This highlights the importance of designing vectors with smaller, dened mutations to fully optimise the biodistribution and toxicity prole of Ad-based vectors.
724. Evaluation of a Porcine Adenovirus 3 Vector as a Post-Exposure Vaccine Candidate
Ami Patel,1,2 Suresk K. Tikoo,3 Gary P. Kobinger.1,2 1 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada; 2Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3Vaccine and Infectious Disease Organization/University of Saskatchewan, Saskatoon, SK, Canada. Experimental adenovirus (Ad) vectors are promising vaccine platforms capable of stimulating robust humoral and cellular immune responses. In particular, human adenovirus 5 (AdHu5) based vaccines have afforded successful protection against emerging pathogens including inuenza, Ebola, and SARS. The generation of rapid protective immune responses against emerging zoonotic pathogens may be advantageous for vaccination and post-exposure treatment of at-risk populations including healthcare and support workers. Unfortunately, a large percentage of the human population has naturally-acquired pre-existing immunity against AdHu5 which can reduce vaccine efcacy and potential for vector readministration. To address this issue novel vector platforms are being developed using rare human and other mammalian adenovirus serotypes with low seroprevalence in humans. The current study evaluates protection and immune responses following vaccination against avian inuenza H5N1 with porcine adenovirus 3 (PAV3) as an alternative vehicle with low seroprevalence in the human population. An optimized expression cassette containing the A/Hanoi/30408/2005 hemagglutinin (HA) gene was inserted into a replication incompetent PAV3 vector by homologous recombination (PAV3-HA). Protection was monitored in BALB/c mice following lethal challenge with homologous H5N1 virus and an AdHu5-HA vaccine was also developed in parallel for comparison. PAV3-HA and AdHu5-HA shared similar protective efcacy against lethal challenge 28-days post-vaccination. Additionally, hemagglutination inhibition (HI) and neutralizing antibody (NAB) responses were comparable for both vaccines, although NAB titers were signicantly higher for PAV3HA at early days post-vaccination. Protection against inuenza is strongly mediated by the humoral response however a good cellular response can accelerate viral clearance. T-cell responses detected by the production of interferon-gamma were signicantly higher for the PAV3-HA vaccine early post-immunization when compared to Adhu5-HA. The generation of fast T and B-cell immune responses suggested that the PAV3 vector may be a good candidate for inducing rapid protective immunity and therefore successful post-exposure vaccination. To address this possibility, mice were immunized with the PAV3-HA vaccine at 5, 8 or 10 days before being submitted to a lethal challenge with H5N1 virus or 1 hour after challenge. Survival and virus titer in the lungs were compared with the AdHu5-HA vaccine. Interestingly, lung homogenate from PAV3-HA vaccinated animals had lower average virus titers on day 3 following challenge. Overall, PAV3-based vectors may offer a promising alternative to traditional AdHu5 vectors for usual prophylaxis vaccination in addition to the rapid induction of protective immune responses after exposure to infectious pathogens.
725. The Knob Domain Replacement Strategy Is Broadly Applicable to a Wide Variety of Adenovirus Fiber Proteins Galina Mikheeva, Natalya Belousova, Victor Krasnykh. Experimental Diagnostic Imaging, The University of Texas M.D. Anderson Cancer Center, Houston, TX.
The inability of adenovirus (Ad) vectors to deliver genes to cells in a target-specic manner remains one of the major limitations of these vectors. Previous successes in Ad targeting were largely limited to vectors derived from human Ad serotype 5 (Ad5). The practical S282
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy