American Society of Hematology

Figure 2

$Figure 2. Human plasma mediates binding of Ad5 to erythrocytes via the complement cascade and CR1. (A) Ad5 was incubated with whole fresh blood and cell fractionation using CPT vacutainers (BD Biosciences) and erythrocyte lysis was performed; the amount of Ad5 associated with each fraction was then quantified by quantitative PCR; N = 4 separate donors, SEM shown. **P < .005. (B) Erythrocytes were isolated and washed before resuspension in either PBS or neutralizing plasma. Erythrocytes were preincubated or not with anti-CAR antibody (RmcB) and then incubated with Ad5; after separation of the liquid (□) and cell fraction (■) by centrifugation, quantification was achieved by quantitative PCR; n = 4, SEM shown, **P < .005. (C) Erythrocytes were thoroughly washed in PBS and resuspended in a variety of plasmas. Ad5 was added and after incubation, the liquid (□) and erythrocyte (■) fractions were separated by centrifugation and analyzed by quantitative PCR for Ad5 genome content (see “Quantitation of Ad5 binding to erythrocytes by real-time (quantitative) PCR”); N = 4, SEM shown. **P < .005. (D) Western blot analysis using anti-C3 antibody was performed to detect the formation of covalent C3-Ad5 adducts: lane 1 indicates Ad5; lane 2, Ad5 + heparin plasma; lane 3, Ad5 + ethylenediaminetetraacetic acid plasma; lane 4, heparin plasma; lane 5, ethylenediaminetetraacetic acid plasma. (E) In human plasma, inhibitors of the binding of complement to CR1 inhibit the association of Ad5 with erythrocytes. Ad5 was added to erythrocytes in plasma in the presence or absence of antibodies against CR1 or C1q or to plasma that had been pretreated with CVF or heat treated to 56°C for 30 minutes. After fractionation of erythrocytes and plasma, Ad5 genome content of each fraction (□ represents plasma; ■, cells) was quantified by quantitative PCR; N = 4, SEM shown. **P < .005. (F) Schematic representing the binding of Ad5 to erythrocytes in PBS or plasma. In PBS, Ad5 binds erythrocytes via CAR but cannot bind via CR1 because of an absence of complement. In neutralizing plasma, Ad5 binds via CR1 but cannot bind to CAR because the epitopes of fiber protein responsible for such binding are covered by neutralizing antibodies. (G) The presence of human erythrocytes in NOD-SCID mice alters Ad5 circulation kinetics. Ad5 was administered intravenously to NOD-SCID mice transplanted with a 10% vol/vol total blood volume of washed human erythrocytes. At defined time points, blood was sampled, separated into plasma and cell fractions, and assayed for Ad5 content by quantitative PCR. Black line/triangle represents dose recovered from the cell fraction of mice treated with human erythrocytes; black dashed line/square, cell fraction from mice that did not receive human erythrocytes; gray line/triangle, plasma fraction from mice that received human erythrocytes; gray dashed line/square, plasma fraction from mice that did not receive human erythrocytes; N = 3, SD shown. **P < .005. (H) The presence of human erythrocytes in NOD-SCID mice inhibits deposition of an intravenously administered Ad5 dose within subcutaneous xenograft tumors. HT29 cells were implanted into NOD-SCID mice and allowed to establish. Mice were injected with Ad5 in the presence or absence of 10% (vol/vol) total blood volume of fresh washed human erythrocytes. After 24 hours, tumors were harvested and assessed for virus genome content. Grubbs outlier test was applied to remove statistical outliers, leaving N = 4, SD shown. *P < .05.$

Human plasma mediates binding of Ad5 to erythrocytes via the complement cascade and CR1. (A) Ad5 was incubated with whole fresh blood and cell fractionation using CPT vacutainers (BD Biosciences) and erythrocyte lysis was performed; the amount of Ad5 associated with each fraction was then quantified by quantitative PCR; N = 4 separate donors, SEM shown. **P < .005. (B) Erythrocytes were isolated and washed before resuspension in either PBS or neutralizing plasma. Erythrocytes were preincubated or not with anti-CAR antibody (RmcB) and then incubated with Ad5; after separation of the liquid (□) and cell fraction (■) by centrifugation, quantification was achieved by quantitative PCR; n = 4, SEM shown, **P < .005. (C) Erythrocytes were thoroughly washed in PBS and resuspended in a variety of plasmas. Ad5 was added and after incubation, the liquid (□) and erythrocyte (■) fractions were separated by centrifugation and analyzed by quantitative PCR for Ad5 genome content (see “Quantitation of Ad5 binding to erythrocytes by real-time (quantitative) PCR”); N = 4, SEM shown. **P < .005. (D) Western blot analysis using anti-C3 antibody was performed to detect the formation of covalent C3-Ad5 adducts: lane 1 indicates Ad5; lane 2, Ad5 + heparin plasma; lane 3, Ad5 + ethylenediaminetetraacetic acid plasma; lane 4, heparin plasma; lane 5, ethylenediaminetetraacetic acid plasma. (E) In human plasma, inhibitors of the binding of complement to CR1 inhibit the association of Ad5 with erythrocytes. Ad5 was added to erythrocytes in plasma in the presence or absence of antibodies against CR1 or C1q or to plasma that had been pretreated with CVF or heat treated to 56°C for 30 minutes. After fractionation of erythrocytes and plasma, Ad5 genome content of each fraction (□ represents plasma; ■, cells) was quantified by quantitative PCR; N = 4, SEM shown. **P < .005. (F) Schematic representing the binding of Ad5 to erythrocytes in PBS or plasma. In PBS, Ad5 binds erythrocytes via CAR but cannot bind via CR1 because of an absence of complement. In neutralizing plasma, Ad5 binds via CR1 but cannot bind to CAR because the epitopes of fiber protein responsible for such binding are covered by neutralizing antibodies. (G) The presence of human erythrocytes in NOD-SCID mice alters Ad5 circulation kinetics. Ad5 was administered intravenously to NOD-SCID mice transplanted with a 10% vol/vol total blood volume of washed human erythrocytes. At defined time points, blood was sampled, separated into plasma and cell fractions, and assayed for Ad5 content by quantitative PCR. Black line/triangle represents dose recovered from the cell fraction of mice treated with human erythrocytes; black dashed line/square, cell fraction from mice that did not receive human erythrocytes; gray line/triangle, plasma fraction from mice that received human erythrocytes; gray dashed line/square, plasma fraction from mice that did not receive human erythrocytes; N = 3, SD shown. **P < .005. (H) The presence of human erythrocytes in NOD-SCID mice inhibits deposition of an intravenously administered Ad5 dose within subcutaneous xenograft tumors. HT29 cells were implanted into NOD-SCID mice and allowed to establish. Mice were injected with Ad5 in the presence or absence of 10% (vol/vol) total blood volume of fresh washed human erythrocytes. After 24 hours, tumors were harvested and assessed for virus genome content. Grubbs outlier test was applied to remove statistical outliers, leaving N = 4, SD shown. *P < .05.

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