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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

Ad binding to human serum components. Alexa Fluor 532-labelled (a+b) Ad5 or (c) Ad5T* (1 × 109 vp) was incubated at room temperature for 90 min in human sera −/+ 40 μg ml−1 Xbp (25 μl final volume). (a) Sera Group A=sensitive to neutralisation in the absence of FX (1 × representative of this group, serum sample #5, shown here), (b) Group B=not sensitive to neutralisation in the absence of FX (1 × representative of this group, serum sample #24 presented here). Samples were diluted in 1.5 ml phosphate-buffered saline and injected into the Nanosight. Each result is representative data from minimum three separate experiments with ~700 completed tracks. Mean±s.e.m. compared by one-way analysis of variance, Tukey's post hoc test, *P<0.05 vs matched control or serum–Xbp conditions.
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fig3: Ad binding to human serum components. Alexa Fluor 532-labelled (a+b) Ad5 or (c) Ad5T* (1 × 109 vp) was incubated at room temperature for 90 min in human sera −/+ 40 μg ml−1 Xbp (25 μl final volume). (a) Sera Group A=sensitive to neutralisation in the absence of FX (1 × representative of this group, serum sample #5, shown here), (b) Group B=not sensitive to neutralisation in the absence of FX (1 × representative of this group, serum sample #24 presented here). Samples were diluted in 1.5 ml phosphate-buffered saline and injected into the Nanosight. Each result is representative data from minimum three separate experiments with ~700 completed tracks. Mean±s.e.m. compared by one-way analysis of variance, Tukey's post hoc test, *P<0.05 vs matched control or serum–Xbp conditions.

Mentions: We next investigated whether FX coating Ad5 was preventing recognition and direct binding of components in the whole human sera, to the viral capsid. We divided our serum samples into two subsets, those in which FX was required to protect the virus from neutralisation (group A) and those in which FX protection was not necessary for cellular transduction (Figure 2). Representative samples from each subset were incubated with Ad5 or Ad5T* in the absence or presence of Xbp and vp size was measured using the NanoSight LM14. There was a significant shift in Ad5 (Figures 3a and b) and Ad5T* (Figure 3c) particle size following incubation with sera from either group indicative of interactions with serum composites. These sera represent a heterogeneous population and some possess low levels of hIgG NAbs capable of binding the virus regardless of FX (Figures 2a and b). However, only in the presence of group A sera did Xbp result in a significant increase in Ad5 particle size compared with incubation with sera alone (Figure 3a). This is indicative of binding of Ad5 by hIgM and classical complement components in the absence of FX.


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)

Ad binding to human serum components. Alexa Fluor 532-labelled (a+b) Ad5 or (c) Ad5T* (1 × 109 vp) was incubated at room temperature for 90 min in human sera −/+ 40 μg ml−1 Xbp (25 μl final volume). (a) Sera Group A=sensitive to neutralisation in the absence of FX (1 × representative of this group, serum sample #5, shown here), (b) Group B=not sensitive to neutralisation in the absence of FX (1 × representative of this group, serum sample #24 presented here). Samples were diluted in 1.5 ml phosphate-buffered saline and injected into the Nanosight. Each result is representative data from minimum three separate experiments with ~700 completed tracks. Mean±s.e.m. compared by one-way analysis of variance, Tukey's post hoc test, *P<0.05 vs matched control or serum–Xbp conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940928&req=5

fig3: Ad binding to human serum components. Alexa Fluor 532-labelled (a+b) Ad5 or (c) Ad5T* (1 × 109 vp) was incubated at room temperature for 90 min in human sera −/+ 40 μg ml−1 Xbp (25 μl final volume). (a) Sera Group A=sensitive to neutralisation in the absence of FX (1 × representative of this group, serum sample #5, shown here), (b) Group B=not sensitive to neutralisation in the absence of FX (1 × representative of this group, serum sample #24 presented here). Samples were diluted in 1.5 ml phosphate-buffered saline and injected into the Nanosight. Each result is representative data from minimum three separate experiments with ~700 completed tracks. Mean±s.e.m. compared by one-way analysis of variance, Tukey's post hoc test, *P<0.05 vs matched control or serum–Xbp conditions.
Mentions: We next investigated whether FX coating Ad5 was preventing recognition and direct binding of components in the whole human sera, to the viral capsid. We divided our serum samples into two subsets, those in which FX was required to protect the virus from neutralisation (group A) and those in which FX protection was not necessary for cellular transduction (Figure 2). Representative samples from each subset were incubated with Ad5 or Ad5T* in the absence or presence of Xbp and vp size was measured using the NanoSight LM14. There was a significant shift in Ad5 (Figures 3a and b) and Ad5T* (Figure 3c) particle size following incubation with sera from either group indicative of interactions with serum composites. These sera represent a heterogeneous population and some possess low levels of hIgG NAbs capable of binding the virus regardless of FX (Figures 2a and b). However, only in the presence of group A sera did Xbp result in a significant increase in Ad5 particle size compared with incubation with sera alone (Figure 3a). This is indicative of binding of Ad5 by hIgM and classical complement components in the absence of FX.

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