Complement activation by recombinant adenoviruses

Abstract

Recombinant adenoviruses are currently the most important vector system in gene therapy. Adenoviruses frequently cause upper respiratory tract infections in humans and anti-adenoviral antibodies are found in 35-70% of the population. Therefore in the majority of potential patients receiving adenoviral gene therapy, the contact of virus particles and blood will lead to the formation of antigen-antibody complexes. These complexes have the ability to induce inflammatory reactions via an activation of the complement system. We have determined the level of C3a (the most reactive complement component) generated in isolated citrate plasma of healthy individuals after challenge with recombinant and wild-type adenoviruses in amounts corresponding to virus blood levels to be expected in patients during adenoviral gene therapy. All plasma samples containing anti-adenoviral antibodies showed a substantial, dose-dependent generation of C3a. A virus plasma level of about 7.5 x 10(9) particles/ml (which was calculated to be the highest blood level reached during clinical trials in the past) induced an average release of about 3000 ng/ml C3a (baseline levels <140 ng/ml). Analyzing the nature of anti-adenoviral antibodies showed, that not only antibodies with neutralizing properties (anti-Ad5), but also non-neutralizing anti-adenoviral antibodies are capable of complement activation. This study suggests that complement activation can be ignored in local low-dose applications of recombinant adenoviruses, but warrants attention after systemic application of large viral quantities. In clinical protocols aiming at systemic virus application, measures for monitoring and controlling the complement system should be included on a regular basis.

Figure 1

Dose-dependency of complement activation in human plasma after stimulation with recombinant serotype 5 adenoviruses in the presence and absence of neutralizing antibodies. Citrate plasma samples of healthy volunteers (n = 4) were incubated with viral particles in four different concentrations (5 × 10⁷–5 × 10¹⁰ particles Ad-lacZ/ml plasma). The reaction was stopped after 90 min at 37°C by addition of EDTA and C3a-desArg levels were determined subsequently. All samples included in this comparison contained anti-adenoviral antibodies (ELISA; 11–702 U/ml), while half of the samples contained neutralizing antibodies (titer 1/256–1/1024) and the second half did not contain neutralising antibodies (titer <1/4). Control experiments were performed with heat-aggregated IgG (HAAG). Average levels of plasma C3a-desArg (n = 3) generated after stimulation with 1/5 vol 1, 2, 5 and 12 mg/ml HAAG. After challenge with 5 and 12 mg/ml HAAG C3a-desArg generation starts to saturate.

Figure 2

Crossreactivity between different adenoviral serotypes. Citrate plasma samples of six probands (anti-adenovirus antibodies in U/ml; IBL-ELISA) were challenged separately with 5 × 10¹⁰ particles/ml wt Ad1, 3, 4, 5 and 9. Ad1 induced the strongest and Ad4 (subgenus E) the weakest C3a release compared with each other serotype.

Figure 3

Evaluation of antibody independent complement activation by serotype 5 adenoviruses. To distinguish antibody-dependent and antibody-independent activation pathways, plasma samples were incubated with adenovirus particles under varying conditions: (a) untreated (control), (b) adenovirus only, (c) pretreatment with EGTA (MgCl₂) (blocks the antibody-dependent activation), (d) depletion of immunoglobulin (Ig) by G-sepharose pretreatment and (e) reconstitution of previously depleted plasma with purified polyvalent human Ig. C3a-desArg release was fully blocked by EGTA pretreament. The increased background levels in Ig-depleted plasma are the result of the G-sepharose treatment. In Ig-depleted plasma, only minimal response was measured after adenovirus challenge, while reconstitution of Ig (20% of physiological levels) reinstalled the previous sensitivity to adenovirus exposure. The outcome illustrates the strict antibody dependence of adenovirus-mediated complement activation in human plasma.