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The relevance of coagulation factor X protection of adenoviruses in human sera.

Duffy MR, Doszpoly A, Turner G, Nicklin SA, Baker AH - Gene Ther. (2016)

Bottom Line: However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples.In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer.In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK.

ABSTRACT
Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5-FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad-FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy.

No MeSH data available.


Related in: MedlinePlus

Screening human sera samples to investigate a protective role of FX. (a) A549 and (b) SKOV3 cells: Ad5 (2 × 1010 vp ml−1) were incubated with media (control) or 25 different human sera −/+40 μg ml−1 Xbp for 30 min at 37 °C. (c) SKOV3 cells: Ad5 or Ad5T* (2 × 1010 vp ml−1) was incubated with media (CON), human or mouse serum −/+ 40 μg ml−1 Xbp for 30 min at 37 °C. Representative human serum samples which did not show a dependence on FX for protection (pooled sera #17, 22, 24) were used in this experiment. Virus suspensions were diluted 200-fold in serum-free media and 100 μl added to cells for 2 h at 37 °C, then replaced with media with 2% fetal calf serum. Transgene expression was quantified ~16 h post transduction and relative light units (RLUs) were normalised to mg total protein. Graphs show transduction as a percentage of control (Ad transduction with media). Media control (*P<0.05) or matched serum–Xbp conditions (#P<0.05) vs serum+Xbp.
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fig2: Screening human sera samples to investigate a protective role of FX. (a) A549 and (b) SKOV3 cells: Ad5 (2 × 1010 vp ml−1) were incubated with media (control) or 25 different human sera −/+40 μg ml−1 Xbp for 30 min at 37 °C. (c) SKOV3 cells: Ad5 or Ad5T* (2 × 1010 vp ml−1) was incubated with media (CON), human or mouse serum −/+ 40 μg ml−1 Xbp for 30 min at 37 °C. Representative human serum samples which did not show a dependence on FX for protection (pooled sera #17, 22, 24) were used in this experiment. Virus suspensions were diluted 200-fold in serum-free media and 100 μl added to cells for 2 h at 37 °C, then replaced with media with 2% fetal calf serum. Transgene expression was quantified ~16 h post transduction and relative light units (RLUs) were normalised to mg total protein. Graphs show transduction as a percentage of control (Ad transduction with media). Media control (*P<0.05) or matched serum–Xbp conditions (#P<0.05) vs serum+Xbp.

Mentions: Next, we investigated whether hIgM and complement components neutralised Ad5 in the absence of FX in whole human sera. A panel of 25 human sera, previously identified in our lab as having low levels of pre-existing neutralising hIgG antibodies (NAbs) to Ad5 (data not shown and some serum samples identified by Parker et al.14 and renumbered for this study), was tested for effects on viral transduction in vitro in the absence or presence of FX. Xbp binds to the FX Gla domain and inhibits its interaction with the virus.1 Owing to the presence of endogenous coagulation factors in the human sera, several samples enhanced Ad5 cellular transduction, an effect significantly reduced by Xbp (Figures 2a and b). The extent to which FX enhanced Ad5 transduction varied, and this can be the result of differences in the endogenous concentrations of FX across the human subsets following blood clotting and serum production and because of altering levels of NAbs. Of the 25 sera examined, in 14 samples (56%), Xbp decreased Ad5 transgene expression to levels significantly below both media controls and serum alone (-Xbp) in A549 cells (Figure 2a). This demonstrated that without the FX protective coat, the virus is neutralised by these sera. Importantly, in the remainder of human samples (44%), Xbp did not decrease Ad5 transduction compared with controls or incubation with serum alone, demonstrating that FX was not required for basal transduction under these conditions. Similar results were observed using SKOV3 cells, although there were some differences amongst the cell lines (4 of the 25 sera caused significant neutralisation compared with media controls and serum alone in only one cell type) (Figure 2b). Previous studies in mice have shown that the ability of IgM to inhibit Ad5 gene transfer is directly related to the antibody titre, with the concentration of murine IgM negatively correlating with transduction.15 Variations in the levels of an individual's natural antibodies may also contribute to differences shown here amongst our human sera samples.


The relevance of coagulation factor X protection of adenoviruses in human sera.

Duffy MR, Doszpoly A, Turner G, Nicklin SA, Baker AH - Gene Ther. (2016)

Screening human sera samples to investigate a protective role of FX. (a) A549 and (b) SKOV3 cells: Ad5 (2 × 1010 vp ml−1) were incubated with media (control) or 25 different human sera −/+40 μg ml−1 Xbp for 30 min at 37 °C. (c) SKOV3 cells: Ad5 or Ad5T* (2 × 1010 vp ml−1) was incubated with media (CON), human or mouse serum −/+ 40 μg ml−1 Xbp for 30 min at 37 °C. Representative human serum samples which did not show a dependence on FX for protection (pooled sera #17, 22, 24) were used in this experiment. Virus suspensions were diluted 200-fold in serum-free media and 100 μl added to cells for 2 h at 37 °C, then replaced with media with 2% fetal calf serum. Transgene expression was quantified ~16 h post transduction and relative light units (RLUs) were normalised to mg total protein. Graphs show transduction as a percentage of control (Ad transduction with media). Media control (*P<0.05) or matched serum–Xbp conditions (#P<0.05) vs serum+Xbp.
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Related In: Results  -  Collection

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fig2: Screening human sera samples to investigate a protective role of FX. (a) A549 and (b) SKOV3 cells: Ad5 (2 × 1010 vp ml−1) were incubated with media (control) or 25 different human sera −/+40 μg ml−1 Xbp for 30 min at 37 °C. (c) SKOV3 cells: Ad5 or Ad5T* (2 × 1010 vp ml−1) was incubated with media (CON), human or mouse serum −/+ 40 μg ml−1 Xbp for 30 min at 37 °C. Representative human serum samples which did not show a dependence on FX for protection (pooled sera #17, 22, 24) were used in this experiment. Virus suspensions were diluted 200-fold in serum-free media and 100 μl added to cells for 2 h at 37 °C, then replaced with media with 2% fetal calf serum. Transgene expression was quantified ~16 h post transduction and relative light units (RLUs) were normalised to mg total protein. Graphs show transduction as a percentage of control (Ad transduction with media). Media control (*P<0.05) or matched serum–Xbp conditions (#P<0.05) vs serum+Xbp.
Mentions: Next, we investigated whether hIgM and complement components neutralised Ad5 in the absence of FX in whole human sera. A panel of 25 human sera, previously identified in our lab as having low levels of pre-existing neutralising hIgG antibodies (NAbs) to Ad5 (data not shown and some serum samples identified by Parker et al.14 and renumbered for this study), was tested for effects on viral transduction in vitro in the absence or presence of FX. Xbp binds to the FX Gla domain and inhibits its interaction with the virus.1 Owing to the presence of endogenous coagulation factors in the human sera, several samples enhanced Ad5 cellular transduction, an effect significantly reduced by Xbp (Figures 2a and b). The extent to which FX enhanced Ad5 transduction varied, and this can be the result of differences in the endogenous concentrations of FX across the human subsets following blood clotting and serum production and because of altering levels of NAbs. Of the 25 sera examined, in 14 samples (56%), Xbp decreased Ad5 transgene expression to levels significantly below both media controls and serum alone (-Xbp) in A549 cells (Figure 2a). This demonstrated that without the FX protective coat, the virus is neutralised by these sera. Importantly, in the remainder of human samples (44%), Xbp did not decrease Ad5 transduction compared with controls or incubation with serum alone, demonstrating that FX was not required for basal transduction under these conditions. Similar results were observed using SKOV3 cells, although there were some differences amongst the cell lines (4 of the 25 sera caused significant neutralisation compared with media controls and serum alone in only one cell type) (Figure 2b). Previous studies in mice have shown that the ability of IgM to inhibit Ad5 gene transfer is directly related to the antibody titre, with the concentration of murine IgM negatively correlating with transduction.15 Variations in the levels of an individual's natural antibodies may also contribute to differences shown here amongst our human sera samples.

Bottom Line: However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples.In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer.In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK.

ABSTRACT
Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5-FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad-FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy.

No MeSH data available.


Related in: MedlinePlus