Structured Review

Affibody ad5 vector
Reported Interactions of <t>Ad5</t> with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].
Ad5 Vector, supplied by Affibody, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors"

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

Journal: Viruses

doi: 10.3390/v2102290

Reported Interactions of Ad5 with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].
Figure Legend Snippet: Reported Interactions of Ad5 with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].

Techniques Used: In Vivo, Binding Assay, Expressing

In Vitro Entry Pathway of Ad5. 1. Ad5 attachment is mediated by binding of the fiber knob to the 46 kDa transmembrane receptor CAR [ 26 – 32 ]. 2. An interaction between the RGD motif with the penton base triggers internalization by clathrin-mediated endocytosis, via ανβ3/5 integrins [ 33 ]. 3. Partial disassembly of the capsid is induced upon acidification of the endosome [ 43 ]. Endosomal escape is modulated through the lytic action of protein VI [ 45 ]. 4. The nucleocapsid-hexon core is translocated to the nuclear pore complex (NPC) along the microtubule network using the microtubule-associated motor, dynein [ 46 , 47 ]. 5. The capsid undergoes its final dissociation event at the nuclear pore complex [ 47 ], allowing the core DNA to extrude into the nucleus for subsequent transcription and replication [ 48 ].
Figure Legend Snippet: In Vitro Entry Pathway of Ad5. 1. Ad5 attachment is mediated by binding of the fiber knob to the 46 kDa transmembrane receptor CAR [ 26 – 32 ]. 2. An interaction between the RGD motif with the penton base triggers internalization by clathrin-mediated endocytosis, via ανβ3/5 integrins [ 33 ]. 3. Partial disassembly of the capsid is induced upon acidification of the endosome [ 43 ]. Endosomal escape is modulated through the lytic action of protein VI [ 45 ]. 4. The nucleocapsid-hexon core is translocated to the nuclear pore complex (NPC) along the microtubule network using the microtubule-associated motor, dynein [ 46 , 47 ]. 5. The capsid undergoes its final dissociation event at the nuclear pore complex [ 47 ], allowing the core DNA to extrude into the nucleus for subsequent transcription and replication [ 48 ].

Techniques Used: In Vitro, Binding Assay

Related Articles

Generated:

Article Title: The Evolution of Adenoviral Vectors through Genetic and Chemical Surface Modifications
Article Snippet: .. Belousova and colleagues have generated an Ad5 vector targeted to human epidermal growth factor receptor 2 (Her2)-positive cells through the incorporation into the fiber of an affibody, which is specific for Her2. .. This re-targeted vector was able to transduce Her2 positive breast cancer cells, along with consistent and significant decrease in transduction of Her2 negative cells in comparison with normal Ad5 [ ].

Binding Assay:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ]. ..

Article Title: A Transductionally Retargeted Adenoviral Vector for Virotherapy of Her2/neu-Expressing Prostate Cancer
Article Snippet: .. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody® molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). .. In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3].

Plasmid Preparation:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ]. ..

Article Title: A Transductionally Retargeted Adenoviral Vector for Virotherapy of Her2/neu-Expressing Prostate Cancer
Article Snippet: .. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody® molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). .. In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3].

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  • 88
    Affibody ad5 vector
    Reported Interactions of <t>Ad5</t> with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].
    Ad5 Vector, supplied by Affibody, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ad5 vector/product/Affibody
    Average 88 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    ad5 vector - by Bioz Stars, 2020-05
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    90
    Affibody ad5
    Increased expression of the F 43S5 11F Her2:7 chimera yields an Ad43 vector that is capable of efficient Her2-specific transduction and is not neutralized by <t>anti-Ad5</t> Abs The western blotting (panel A ) and SDS-PAGE (panel B ) of CsCl-purified virions (10 10 VP/lane) of Ad43TL.F 43S5 11F Her2:7 amplified in 293F 43S5 11F Her2:7 cells (lane 1), and Ad43TL (lane 2). FC, the F 43S5 11F Her2:7 fiber chimera. The images shown in panel 7A and 7B have been cropped and regrouped. The gene transfer to 293/Her2 and 293 cells (panel C ), and tumor cell lines expressing Her2 (panel D ). The Zher2-7 affibody was used as a Her2-specific inhibitor (panel D only). Ad43TL.F 43S5 11F Her2:7 , 1; Ad43TL, 2. The Her2-negative MDA-MB-231 cells, a parental line for Her2-expressing MDA-MB-231/Her2 cells, are shown as a control. ( E ) In vitro vector neutralization assay. The sera obtained from naïve or Ad5-immunized mice were added to 293/Her2 cells prior to transduction with Ad5TL or Ad43TL.F 43S5 11F Her2:7 vectors. The levels of transgene expression in transduced cells relative to expression level seen in the absence of serum are shown. The sera dilution factors are shown below the graphs. NS, no serum. In panels C, D, and E, data are shown as the mean of three replicates; error bars show the standard deviations. * p
    Ad5, supplied by Affibody, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ad5/product/Affibody
    Average 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
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    85
    Affibody anti ad5 fiber tail mab
    sAd24 fiber-derived targeting chimera forms stable trimers and binds to cell-associated Her2. (A) Western blotting of lysates of 293T cells transiently expressing the fiber constructs. Lanes 1 and 2, wt <t>Ad5</t> fiber; lanes 3 and 4, F sAd24 11F Her2:7 , lanes
    Anti Ad5 Fiber Tail Mab, supplied by Affibody, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Reported Interactions of Ad5 with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].

    Journal: Viruses

    Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    doi: 10.3390/v2102290

    Figure Lengend Snippet: Reported Interactions of Ad5 with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].

    Article Snippet: Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ].

    Techniques: In Vivo, Binding Assay, Expressing

    In Vitro Entry Pathway of Ad5. 1. Ad5 attachment is mediated by binding of the fiber knob to the 46 kDa transmembrane receptor CAR [ 26 – 32 ]. 2. An interaction between the RGD motif with the penton base triggers internalization by clathrin-mediated endocytosis, via ανβ3/5 integrins [ 33 ]. 3. Partial disassembly of the capsid is induced upon acidification of the endosome [ 43 ]. Endosomal escape is modulated through the lytic action of protein VI [ 45 ]. 4. The nucleocapsid-hexon core is translocated to the nuclear pore complex (NPC) along the microtubule network using the microtubule-associated motor, dynein [ 46 , 47 ]. 5. The capsid undergoes its final dissociation event at the nuclear pore complex [ 47 ], allowing the core DNA to extrude into the nucleus for subsequent transcription and replication [ 48 ].

    Journal: Viruses

    Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    doi: 10.3390/v2102290

    Figure Lengend Snippet: In Vitro Entry Pathway of Ad5. 1. Ad5 attachment is mediated by binding of the fiber knob to the 46 kDa transmembrane receptor CAR [ 26 – 32 ]. 2. An interaction between the RGD motif with the penton base triggers internalization by clathrin-mediated endocytosis, via ανβ3/5 integrins [ 33 ]. 3. Partial disassembly of the capsid is induced upon acidification of the endosome [ 43 ]. Endosomal escape is modulated through the lytic action of protein VI [ 45 ]. 4. The nucleocapsid-hexon core is translocated to the nuclear pore complex (NPC) along the microtubule network using the microtubule-associated motor, dynein [ 46 , 47 ]. 5. The capsid undergoes its final dissociation event at the nuclear pore complex [ 47 ], allowing the core DNA to extrude into the nucleus for subsequent transcription and replication [ 48 ].

    Article Snippet: Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ].

    Techniques: In Vitro, Binding Assay

    Increased expression of the F 43S5 11F Her2:7 chimera yields an Ad43 vector that is capable of efficient Her2-specific transduction and is not neutralized by anti-Ad5 Abs The western blotting (panel A ) and SDS-PAGE (panel B ) of CsCl-purified virions (10 10 VP/lane) of Ad43TL.F 43S5 11F Her2:7 amplified in 293F 43S5 11F Her2:7 cells (lane 1), and Ad43TL (lane 2). FC, the F 43S5 11F Her2:7 fiber chimera. The images shown in panel 7A and 7B have been cropped and regrouped. The gene transfer to 293/Her2 and 293 cells (panel C ), and tumor cell lines expressing Her2 (panel D ). The Zher2-7 affibody was used as a Her2-specific inhibitor (panel D only). Ad43TL.F 43S5 11F Her2:7 , 1; Ad43TL, 2. The Her2-negative MDA-MB-231 cells, a parental line for Her2-expressing MDA-MB-231/Her2 cells, are shown as a control. ( E ) In vitro vector neutralization assay. The sera obtained from naïve or Ad5-immunized mice were added to 293/Her2 cells prior to transduction with Ad5TL or Ad43TL.F 43S5 11F Her2:7 vectors. The levels of transgene expression in transduced cells relative to expression level seen in the absence of serum are shown. The sera dilution factors are shown below the graphs. NS, no serum. In panels C, D, and E, data are shown as the mean of three replicates; error bars show the standard deviations. * p

    Journal: Oncotarget

    Article Title: Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43

    doi: 10.18632/oncotarget.10800

    Figure Lengend Snippet: Increased expression of the F 43S5 11F Her2:7 chimera yields an Ad43 vector that is capable of efficient Her2-specific transduction and is not neutralized by anti-Ad5 Abs The western blotting (panel A ) and SDS-PAGE (panel B ) of CsCl-purified virions (10 10 VP/lane) of Ad43TL.F 43S5 11F Her2:7 amplified in 293F 43S5 11F Her2:7 cells (lane 1), and Ad43TL (lane 2). FC, the F 43S5 11F Her2:7 fiber chimera. The images shown in panel 7A and 7B have been cropped and regrouped. The gene transfer to 293/Her2 and 293 cells (panel C ), and tumor cell lines expressing Her2 (panel D ). The Zher2-7 affibody was used as a Her2-specific inhibitor (panel D only). Ad43TL.F 43S5 11F Her2:7 , 1; Ad43TL, 2. The Her2-negative MDA-MB-231 cells, a parental line for Her2-expressing MDA-MB-231/Her2 cells, are shown as a control. ( E ) In vitro vector neutralization assay. The sera obtained from naïve or Ad5-immunized mice were added to 293/Her2 cells prior to transduction with Ad5TL or Ad43TL.F 43S5 11F Her2:7 vectors. The levels of transgene expression in transduced cells relative to expression level seen in the absence of serum are shown. The sera dilution factors are shown below the graphs. NS, no serum. In panels C, D, and E, data are shown as the mean of three replicates; error bars show the standard deviations. * p

    Article Snippet: Applying the fiber knob replacement targeting approach [ ] to Ad43 in this study revealed some of the limitations of this strategy: in contrast to Ad5 and sAd24 previously targeted using this strategy [ , ], Ad43 could not be targeted to Her2 by simple replacement of its knob domain with the fibritin-affibody fusion.

    Techniques: Expressing, Plasmid Preparation, Transduction, Western Blot, SDS Page, Purification, Amplification, Multiple Displacement Amplification, In Vitro, Neutralization, Mouse Assay

    Lack of interaction between Ad43 virions and FX results in minimal hepatic transduction, but does not affect the vector uptake by the liver ( A ) Surface plasmon resonance analysis of FX interaction with Ad43 and Ad5. Sensorgrams obtained by probing chip-immobilized Ad virions with soluble FX are shown in duplicates, each curve corresponding to a separate run. Serial two-fold dilutions of FX covered a range of concentrations from 0.4 μg/ml (shown in light blue) to 25 μg/ml (shown in red). The calculated K D for Ad5-FX interaction is 1.50 × 10 −9 M. ( B ) Overlays of the whole-body bioluminescent and X-ray images of groups of mice ( n = 3) intravenously injected with Ad5TL and Ad43TL vectors (4 × 10 10 viral particles [VP] each) acquired 48 h after injections. The regions of interest that outline the livers are shown with red frames. The levels of bioluminescence measured in these regions of interest (total flux, photon/s) are shown below the images of mice. The pseudo-colour scale shows the radiance in photons/second/centimeter 2 /radian 2 . ( C ) The activity of the Fluc reporter measured in the homogenates of organs collected from Ad-injected mice 72 h after vectors injections. Each bar represents an average signal intensity in relative light units (RLU) per entire organ. Error bars indicate the standard deviations for a group of three animals; the measurements were taken in duplicates. The Fluc activities in PBS-injected mice were (in RLU/organ): 1.9 × 10 7 (liver), 5 × 10 6 (spleen), 9.9 × 10 5 (lung), 1.2 × 10 6 (heart), 3.4 × 10 6 (kidney). * p

    Journal: Oncotarget

    Article Title: Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43

    doi: 10.18632/oncotarget.10800

    Figure Lengend Snippet: Lack of interaction between Ad43 virions and FX results in minimal hepatic transduction, but does not affect the vector uptake by the liver ( A ) Surface plasmon resonance analysis of FX interaction with Ad43 and Ad5. Sensorgrams obtained by probing chip-immobilized Ad virions with soluble FX are shown in duplicates, each curve corresponding to a separate run. Serial two-fold dilutions of FX covered a range of concentrations from 0.4 μg/ml (shown in light blue) to 25 μg/ml (shown in red). The calculated K D for Ad5-FX interaction is 1.50 × 10 −9 M. ( B ) Overlays of the whole-body bioluminescent and X-ray images of groups of mice ( n = 3) intravenously injected with Ad5TL and Ad43TL vectors (4 × 10 10 viral particles [VP] each) acquired 48 h after injections. The regions of interest that outline the livers are shown with red frames. The levels of bioluminescence measured in these regions of interest (total flux, photon/s) are shown below the images of mice. The pseudo-colour scale shows the radiance in photons/second/centimeter 2 /radian 2 . ( C ) The activity of the Fluc reporter measured in the homogenates of organs collected from Ad-injected mice 72 h after vectors injections. Each bar represents an average signal intensity in relative light units (RLU) per entire organ. Error bars indicate the standard deviations for a group of three animals; the measurements were taken in duplicates. The Fluc activities in PBS-injected mice were (in RLU/organ): 1.9 × 10 7 (liver), 5 × 10 6 (spleen), 9.9 × 10 5 (lung), 1.2 × 10 6 (heart), 3.4 × 10 6 (kidney). * p

    Article Snippet: Applying the fiber knob replacement targeting approach [ ] to Ad43 in this study revealed some of the limitations of this strategy: in contrast to Ad5 and sAd24 previously targeted using this strategy [ , ], Ad43 could not be targeted to Her2 by simple replacement of its knob domain with the fibritin-affibody fusion.

    Techniques: Transduction, Plasmid Preparation, SPR Assay, Chromatin Immunoprecipitation, Mouse Assay, Injection, Activity Assay

    Ad43 fiber-derived Her2-targeting chimeras form trimers and bind to Her2 ( A ) The structure of the Ad43 fiber and the Ad43 fiber-derived chimeras. The Ad43 fiber consists of the tail, shaft, and knob domains. The Ad43 fiber shaft is short (8 repeats) and it lacks a TTVS hinge motif, whose presence in the third pseudo-repeat of the long Ad5 fiber shaft (22 repeats) make the Ad5 fiber flexible. In all chimeras, the tail domain was left intact, whereas the knob domain was replaced with the trimerization domain of phage T4 fibritin protein and a Her2-targeting affibody, Zher2:7. The F 43 11F Her2:7 chimera contains an unmodified Ad43 fiber shaft. To make the chimeras flexible, in F 43M1 11F Her2:7 , the entire third repeat of the Ad43 fiber shaft was replaced with the third repeat of the Ad5 fiber shaft and in F 43M2 11F Her2:7 , the TTVS hinge was inserted into the Ad43 fiber's third repeat. To make the chimera flexible and to simultaneously elongate it, the Ad43 fiber shaft in F 43S5 11F Her2:7 was replaced with the Ad5 fiber shaft. The TTVS hinge is shown in bold; the amino acids in the Ad5 shaft third repeat that differ from those in Ad43 are underlined. Western blot of boiled (panel B ) and non-boiled (panel C ) lysates of 293T cells transiently expressing the fiber chimeras: F 43 11F Her2:7 (lane 2), F 43M1 11F Her2:7 (lane 3), F 43M2 11F Her2:7 (lane 4), and F 43S5 11F Her2:7 (lane 5); the wt Ad43 fiber is in lane 1. (Here and in other panels showing the results of western blotting and SDS-PAGE, lane S shows protein standards with the molecular masses in kDa). All the chimeras formed trimers but were expressed at lower levels than was the wt Ad43 fiber. The predicted molecular masses of monomeric wt Ad43 fiber, F 43 11F Her2:7 , F 43M1 11F Her2:7 , F 43M2 11F Her2:7 , and F 43S5 11F Her2:7 are 39.4, 36.8, 37.3, 37.2, 59.4 kDa, respectively. The image shown in panel 4C has been cropped and regrouped; the lane showing protein standards was digitally enhanced. ( D ) Flow cytometry of Her2-positive and negative cells probed with the designed fiber chimeras. The protein probes are: wt Ad43 fiber (control), 1; F 43 11F Her2:7 , 2; F 43M1 11F Her2:7 , 3; F 43M2 11F Her2:7 , 4; F 43S5 11F Her2:7 , 5; lysate of mock transfected 293T cells, M.

    Journal: Oncotarget

    Article Title: Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43

    doi: 10.18632/oncotarget.10800

    Figure Lengend Snippet: Ad43 fiber-derived Her2-targeting chimeras form trimers and bind to Her2 ( A ) The structure of the Ad43 fiber and the Ad43 fiber-derived chimeras. The Ad43 fiber consists of the tail, shaft, and knob domains. The Ad43 fiber shaft is short (8 repeats) and it lacks a TTVS hinge motif, whose presence in the third pseudo-repeat of the long Ad5 fiber shaft (22 repeats) make the Ad5 fiber flexible. In all chimeras, the tail domain was left intact, whereas the knob domain was replaced with the trimerization domain of phage T4 fibritin protein and a Her2-targeting affibody, Zher2:7. The F 43 11F Her2:7 chimera contains an unmodified Ad43 fiber shaft. To make the chimeras flexible, in F 43M1 11F Her2:7 , the entire third repeat of the Ad43 fiber shaft was replaced with the third repeat of the Ad5 fiber shaft and in F 43M2 11F Her2:7 , the TTVS hinge was inserted into the Ad43 fiber's third repeat. To make the chimera flexible and to simultaneously elongate it, the Ad43 fiber shaft in F 43S5 11F Her2:7 was replaced with the Ad5 fiber shaft. The TTVS hinge is shown in bold; the amino acids in the Ad5 shaft third repeat that differ from those in Ad43 are underlined. Western blot of boiled (panel B ) and non-boiled (panel C ) lysates of 293T cells transiently expressing the fiber chimeras: F 43 11F Her2:7 (lane 2), F 43M1 11F Her2:7 (lane 3), F 43M2 11F Her2:7 (lane 4), and F 43S5 11F Her2:7 (lane 5); the wt Ad43 fiber is in lane 1. (Here and in other panels showing the results of western blotting and SDS-PAGE, lane S shows protein standards with the molecular masses in kDa). All the chimeras formed trimers but were expressed at lower levels than was the wt Ad43 fiber. The predicted molecular masses of monomeric wt Ad43 fiber, F 43 11F Her2:7 , F 43M1 11F Her2:7 , F 43M2 11F Her2:7 , and F 43S5 11F Her2:7 are 39.4, 36.8, 37.3, 37.2, 59.4 kDa, respectively. The image shown in panel 4C has been cropped and regrouped; the lane showing protein standards was digitally enhanced. ( D ) Flow cytometry of Her2-positive and negative cells probed with the designed fiber chimeras. The protein probes are: wt Ad43 fiber (control), 1; F 43 11F Her2:7 , 2; F 43M1 11F Her2:7 , 3; F 43M2 11F Her2:7 , 4; F 43S5 11F Her2:7 , 5; lysate of mock transfected 293T cells, M.

    Article Snippet: Applying the fiber knob replacement targeting approach [ ] to Ad43 in this study revealed some of the limitations of this strategy: in contrast to Ad5 and sAd24 previously targeted using this strategy [ , ], Ad43 could not be targeted to Her2 by simple replacement of its knob domain with the fibritin-affibody fusion.

    Techniques: Derivative Assay, Western Blot, Expressing, SDS Page, Flow Cytometry, Cytometry, Transfection

    Alignment of Ad43 hexon HVRs 2, 3, 5, and 7 with HVRs of FX-binding hexons The Ad5 hexon amino acids residues in HVR5 and HVR7 involved in binding FX [ 10 ] are highlighted in gray; none of them is present in the Ad43 hexon. Shown in bold red are the TET tripeptide (in the Ad5 hexon's HVR7) previously implicated in FX binding [ 5 ] and its homologue, TDT, whose presence in HVRs of other Ads hexons correlates strongly with reported binding to FX. Of the human Ad serotypes whose binding to FX was tested and confirmed [ 5 ], seven contain TDT in their hexons. Six of these Ads are shown below the dashed line.

    Journal: Oncotarget

    Article Title: Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43

    doi: 10.18632/oncotarget.10800

    Figure Lengend Snippet: Alignment of Ad43 hexon HVRs 2, 3, 5, and 7 with HVRs of FX-binding hexons The Ad5 hexon amino acids residues in HVR5 and HVR7 involved in binding FX [ 10 ] are highlighted in gray; none of them is present in the Ad43 hexon. Shown in bold red are the TET tripeptide (in the Ad5 hexon's HVR7) previously implicated in FX binding [ 5 ] and its homologue, TDT, whose presence in HVRs of other Ads hexons correlates strongly with reported binding to FX. Of the human Ad serotypes whose binding to FX was tested and confirmed [ 5 ], seven contain TDT in their hexons. Six of these Ads are shown below the dashed line.

    Article Snippet: Applying the fiber knob replacement targeting approach [ ] to Ad43 in this study revealed some of the limitations of this strategy: in contrast to Ad5 and sAd24 previously targeted using this strategy [ , ], Ad43 could not be targeted to Her2 by simple replacement of its knob domain with the fibritin-affibody fusion.

    Techniques: Binding Assay

    sAd24 fiber-derived targeting chimera forms stable trimers and binds to cell-associated Her2. (A) Western blotting of lysates of 293T cells transiently expressing the fiber constructs. Lanes 1 and 2, wt Ad5 fiber; lanes 3 and 4, F sAd24 11F Her2:7 , lanes

    Journal: Journal of Virology

    Article Title: Development of a Targeted Gene Vector Platform Based on Simian Adenovirus Serotype 24 ▿

    doi: 10.1128/JVI.02425-09

    Figure Lengend Snippet: sAd24 fiber-derived targeting chimera forms stable trimers and binds to cell-associated Her2. (A) Western blotting of lysates of 293T cells transiently expressing the fiber constructs. Lanes 1 and 2, wt Ad5 fiber; lanes 3 and 4, F sAd24 11F Her2:7 , lanes

    Article Snippet: When used in parallel with anti-Ad5 fiber tail MAb to detect the cell-attached Ad5 fiber-derived chimera, the 5E1 MAb produced a much weaker signal (unpublished data), perhaps because of steric hindrance caused by the proximity of its epitope to the affibody-Her2 interface.

    Techniques: Derivative Assay, Western Blot, Expressing, Construct

    Ad5 and sAd24 virions show similar patterns of in vivo distribution. Shown are the copy numbers of Ad5 and sAd24 viral genomes per organ detected by qPCR in isolated murine organs at 1 h and 24 h after systemic administration of the vectors. Error bars

    Journal: Journal of Virology

    Article Title: Development of a Targeted Gene Vector Platform Based on Simian Adenovirus Serotype 24 ▿

    doi: 10.1128/JVI.02425-09

    Figure Lengend Snippet: Ad5 and sAd24 virions show similar patterns of in vivo distribution. Shown are the copy numbers of Ad5 and sAd24 viral genomes per organ detected by qPCR in isolated murine organs at 1 h and 24 h after systemic administration of the vectors. Error bars

    Article Snippet: When used in parallel with anti-Ad5 fiber tail MAb to detect the cell-attached Ad5 fiber-derived chimera, the 5E1 MAb produced a much weaker signal (unpublished data), perhaps because of steric hindrance caused by the proximity of its epitope to the affibody-Her2 interface.

    Techniques: In Vivo, Real-time Polymerase Chain Reaction, Isolation

    Instability of FX-sAd24 complexes results in reduced liver transduction by sAd24-derived vector. (A) The interactions of Ad5 and sAd24 virions with FX were analyzed using surface plasmon resonance. Viral particles bound to the biosensor chip (Ad5 at a

    Journal: Journal of Virology

    Article Title: Development of a Targeted Gene Vector Platform Based on Simian Adenovirus Serotype 24 ▿

    doi: 10.1128/JVI.02425-09

    Figure Lengend Snippet: Instability of FX-sAd24 complexes results in reduced liver transduction by sAd24-derived vector. (A) The interactions of Ad5 and sAd24 virions with FX were analyzed using surface plasmon resonance. Viral particles bound to the biosensor chip (Ad5 at a

    Article Snippet: When used in parallel with anti-Ad5 fiber tail MAb to detect the cell-attached Ad5 fiber-derived chimera, the 5E1 MAb produced a much weaker signal (unpublished data), perhaps because of steric hindrance caused by the proximity of its epitope to the affibody-Her2 interface.

    Techniques: Transduction, Derivative Assay, Plasmid Preparation, SPR Assay, Chromatin Immunoprecipitation

    Comparison of cytokine release in response to intravenous injections of Ad5 and sAd24. Concentrations of IL-6, MCP-1, TNF, and IFN-γ in plasma samples collected from mice at 1 h, 6 h, or 24 h after injection with either Ad5- or sAd24-derived vectors

    Journal: Journal of Virology

    Article Title: Development of a Targeted Gene Vector Platform Based on Simian Adenovirus Serotype 24 ▿

    doi: 10.1128/JVI.02425-09

    Figure Lengend Snippet: Comparison of cytokine release in response to intravenous injections of Ad5 and sAd24. Concentrations of IL-6, MCP-1, TNF, and IFN-γ in plasma samples collected from mice at 1 h, 6 h, or 24 h after injection with either Ad5- or sAd24-derived vectors

    Article Snippet: When used in parallel with anti-Ad5 fiber tail MAb to detect the cell-attached Ad5 fiber-derived chimera, the 5E1 MAb produced a much weaker signal (unpublished data), perhaps because of steric hindrance caused by the proximity of its epitope to the affibody-Her2 interface.

    Techniques: Mouse Assay, Injection, Derivative Assay