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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

Effects on human complement activation. Human sera was incubated with Ad (5 × 1010 vp ml−1) in the presence or absence of 40 μg ml−1 Xbp for 90 min at 37 °C. (a) Human sera with high pre-existing neutralising IgG titres (high NAbs) and sera samples with low IgG titres which exhibited a dependence on FX for protection (low NAbs Group A), (b) FX-depleted human plasma (Quadratech Diagnostics, UK) and (c) C1q depleted human sera were used. C3a-desArg levels were quantified by ELISA. Graphs show transduction as a percentage of serum+phosphate-buffered saline (PBS). *P<0.05 vs matched PBS or Ad5.
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fig4: Effects on human complement activation. Human sera was incubated with Ad (5 × 1010 vp ml−1) in the presence or absence of 40 μg ml−1 Xbp for 90 min at 37 °C. (a) Human sera with high pre-existing neutralising IgG titres (high NAbs) and sera samples with low IgG titres which exhibited a dependence on FX for protection (low NAbs Group A), (b) FX-depleted human plasma (Quadratech Diagnostics, UK) and (c) C1q depleted human sera were used. C3a-desArg levels were quantified by ELISA. Graphs show transduction as a percentage of serum+phosphate-buffered saline (PBS). *P<0.05 vs matched PBS or Ad5.

Mentions: We next studied the early signalling effects of hIgM binding to the virus and the activation of complement pathways. C1q is the recognition component of the classical complement cascade which can bind IgG or IgM leading to the formation of C4b2a convertase, which cleaves C3 to generate C3a.16 C3a is very short lived and is rapidly cleaved in serum to the more stable product C3a-desArg, hence C3a-desArg is commonly used as a measure of complement activation. We observed increased levels of C3a-desArg in both human sera with high and low pre-existing NAbs (source of serum samples Parker et al.14) following incubation with Ad5, and this was further significantly increased by adding Xbp (Figure 4a, Supplementary Figure 1). The enhanced C3-desArg signal in the absence of FX vs its presence suggests that the ability of Ad5 to induce C3-desArg is not solely reliant on FX, however, FX coating does impact upon the extent of complement-induced immunogenicity. In FX-depleted plasma, Ad5 caused comparable levels of C3a-desArg regardless of Xbp (Figure 4b). C3a and C3a-desArg are common components of classical, lectin and alternative complement pathways, whilst C1q is selectively involved in the classical system.17 In human serum devoid of C1q, no significant increase in C3-desArg was observed with Ad5 in the presence or absence of FX (Figure 4c). These data thereby indicate C1q-mediated C3 activation (that is, classical complement) occurs in the presence of FX, but the levels of C3a-desArg induction are significantly increased when FX is absent, in the human sera samples tested. This suggests that the human pathway is more complex than that observed in mice, in which no C3a activation occurs in the absence of FX,2, 8 and having no prior exposure to Ad5, the animals contain no pre-existing NAbs.


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)

Effects on human complement activation. Human sera was incubated with Ad (5 × 1010 vp ml−1) in the presence or absence of 40 μg ml−1 Xbp for 90 min at 37 °C. (a) Human sera with high pre-existing neutralising IgG titres (high NAbs) and sera samples with low IgG titres which exhibited a dependence on FX for protection (low NAbs Group A), (b) FX-depleted human plasma (Quadratech Diagnostics, UK) and (c) C1q depleted human sera were used. C3a-desArg levels were quantified by ELISA. Graphs show transduction as a percentage of serum+phosphate-buffered saline (PBS). *P<0.05 vs matched PBS or Ad5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Effects on human complement activation. Human sera was incubated with Ad (5 × 1010 vp ml−1) in the presence or absence of 40 μg ml−1 Xbp for 90 min at 37 °C. (a) Human sera with high pre-existing neutralising IgG titres (high NAbs) and sera samples with low IgG titres which exhibited a dependence on FX for protection (low NAbs Group A), (b) FX-depleted human plasma (Quadratech Diagnostics, UK) and (c) C1q depleted human sera were used. C3a-desArg levels were quantified by ELISA. Graphs show transduction as a percentage of serum+phosphate-buffered saline (PBS). *P<0.05 vs matched PBS or Ad5.
Mentions: We next studied the early signalling effects of hIgM binding to the virus and the activation of complement pathways. C1q is the recognition component of the classical complement cascade which can bind IgG or IgM leading to the formation of C4b2a convertase, which cleaves C3 to generate C3a.16 C3a is very short lived and is rapidly cleaved in serum to the more stable product C3a-desArg, hence C3a-desArg is commonly used as a measure of complement activation. We observed increased levels of C3a-desArg in both human sera with high and low pre-existing NAbs (source of serum samples Parker et al.14) following incubation with Ad5, and this was further significantly increased by adding Xbp (Figure 4a, Supplementary Figure 1). The enhanced C3-desArg signal in the absence of FX vs its presence suggests that the ability of Ad5 to induce C3-desArg is not solely reliant on FX, however, FX coating does impact upon the extent of complement-induced immunogenicity. In FX-depleted plasma, Ad5 caused comparable levels of C3a-desArg regardless of Xbp (Figure 4b). C3a and C3a-desArg are common components of classical, lectin and alternative complement pathways, whilst C1q is selectively involved in the classical system.17 In human serum devoid of C1q, no significant increase in C3-desArg was observed with Ad5 in the presence or absence of FX (Figure 4c). These data thereby indicate C1q-mediated C3 activation (that is, classical complement) occurs in the presence of FX, but the levels of C3a-desArg induction are significantly increased when FX is absent, in the human sera samples tested. This suggests that the human pathway is more complex than that observed in mice, in which no C3a activation occurs in the absence of FX,2, 8 and having no prior exposure to Ad5, the animals contain no pre-existing NAbs.

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