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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

Ad5 binds hIgM in a FX-dependent manner. Alexa Fluor 532-labelled Ad5 or Ad5T* vectors (1 × 109 vp) were incubated at room temperature for 90 min in phosphate-buffered saline (PBS) (10 μl final volume) with physiologically relevant concentrations of (a) hIgM (100 μg ml−1) or (b) human IgG (10 μg ml−1) in the presence or absence of human FX (5 μg ml−1), then diluted in 1.5 ml PBS and injected into the Nanosight. Each result is representative data from a minimum of three separate experiments with ~300 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 hIgM conditions.
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fig1: Ad5 binds hIgM in a FX-dependent manner. Alexa Fluor 532-labelled Ad5 or Ad5T* vectors (1 × 109 vp) were incubated at room temperature for 90 min in phosphate-buffered saline (PBS) (10 μl final volume) with physiologically relevant concentrations of (a) hIgM (100 μg ml−1) or (b) human IgG (10 μg ml−1) in the presence or absence of human FX (5 μg ml−1), then diluted in 1.5 ml PBS and injected into the Nanosight. Each result is representative data from a minimum of three separate experiments with ~300 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 hIgM conditions.

Mentions: The ability of FX to bind to the Ad5 HVRs, thereby preventing recognition and attack by murine natural antibodies and complement in mice has been documented. To study the importance of this pathway in humans, we began by investigating whether human IgM (hIgM) bound to Ad5 directly and whether this was influenced by human FX. We incubated purified hIgM with Alexa Fluor 532-labelled Ad5 or the FX-binding deficient version Ad5T* (point mutations within the hexon HVR5 and HVR7(ref. 11)) in the absence or presence of FX and analysed alterations in virus particle (vp) size using the NanoSight LM14 (NanoSight, Malvern, UK). Binding of the free proteins to the fluorescently labelled virus correlates directly with an increase in size. The NanoSight LM14 and associated NTA software enables visualisation of nanoparticles in solution, robust particle-by-particle size measurements and quantification based on principles of Brownian motion and fluorescence light scattering.12 The lower size detection limit for the NanoSight LM14 is 10 nm, and whilst a hIgM pentamer is ~30 nm, FX is 9 nm and thus not detectable via this method.12, 13 A significant increase in particle size (~35 nm) was observed in the presence of hIgM, demonstrating a direct interaction with Ad5 (Figure 1a). Importantly, this interaction was inhibited when Ad5 was co-incubated with FX, showing the ability of FX to hinder direct binding of hIgM to Ad5. hIgM also bound the non-FX-binder Ad5T* (Figure 1a), demonstrating that the amino acid residues of the virus responsible for interacting with FX are not required for the hIgM:Ad5 interaction, and as expected, this was unaffected by FX. FX did not interfere with the binding of purified hIgG to Ad5 (Figure 1b).


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)

Ad5 binds hIgM in a FX-dependent manner. Alexa Fluor 532-labelled Ad5 or Ad5T* vectors (1 × 109 vp) were incubated at room temperature for 90 min in phosphate-buffered saline (PBS) (10 μl final volume) with physiologically relevant concentrations of (a) hIgM (100 μg ml−1) or (b) human IgG (10 μg ml−1) in the presence or absence of human FX (5 μg ml−1), then diluted in 1.5 ml PBS and injected into the Nanosight. Each result is representative data from a minimum of three separate experiments with ~300 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 hIgM conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Ad5 binds hIgM in a FX-dependent manner. Alexa Fluor 532-labelled Ad5 or Ad5T* vectors (1 × 109 vp) were incubated at room temperature for 90 min in phosphate-buffered saline (PBS) (10 μl final volume) with physiologically relevant concentrations of (a) hIgM (100 μg ml−1) or (b) human IgG (10 μg ml−1) in the presence or absence of human FX (5 μg ml−1), then diluted in 1.5 ml PBS and injected into the Nanosight. Each result is representative data from a minimum of three separate experiments with ~300 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 hIgM conditions.
Mentions: The ability of FX to bind to the Ad5 HVRs, thereby preventing recognition and attack by murine natural antibodies and complement in mice has been documented. To study the importance of this pathway in humans, we began by investigating whether human IgM (hIgM) bound to Ad5 directly and whether this was influenced by human FX. We incubated purified hIgM with Alexa Fluor 532-labelled Ad5 or the FX-binding deficient version Ad5T* (point mutations within the hexon HVR5 and HVR7(ref. 11)) in the absence or presence of FX and analysed alterations in virus particle (vp) size using the NanoSight LM14 (NanoSight, Malvern, UK). Binding of the free proteins to the fluorescently labelled virus correlates directly with an increase in size. The NanoSight LM14 and associated NTA software enables visualisation of nanoparticles in solution, robust particle-by-particle size measurements and quantification based on principles of Brownian motion and fluorescence light scattering.12 The lower size detection limit for the NanoSight LM14 is 10 nm, and whilst a hIgM pentamer is ~30 nm, FX is 9 nm and thus not detectable via this method.12, 13 A significant increase in particle size (~35 nm) was observed in the presence of hIgM, demonstrating a direct interaction with Ad5 (Figure 1a). Importantly, this interaction was inhibited when Ad5 was co-incubated with FX, showing the ability of FX to hinder direct binding of hIgM to Ad5. hIgM also bound the non-FX-binder Ad5T* (Figure 1a), demonstrating that the amino acid residues of the virus responsible for interacting with FX are not required for the hIgM:Ad5 interaction, and as expected, this was unaffected by FX. FX did not interfere with the binding of purified hIgG to Ad5 (Figure 1b).

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