IdeS: a bacterial proteolytic enzyme with therapeutic potential

Abstract

Background: IdeS, a proteinase from Streptococcus pyogenes, cleaves immunoglobulin (Ig)G antibodies with a unique degree of specificity. Pathogenic IgG antibodies constitute an important clinical problem contributing to the pathogenesis of a number of autoimmune conditions and acute transplant rejection. To be able to effectively remove such antibodies is therefore an important clinical challenge.

Methodology/principal findings: IdeS was found to specifically and efficiently cleave IgG in human blood in vitro (20 microg of IdeS caused a complete degradation of IgG in one ml of human whole blood in 15 minutes) and to clear IgG from the blood stream of rabbits in vivo (no IgG was detected six hours following an intravenous injection of 5 mg of IdeS) without any side effects. In a mouse model of immune thrombocytopenic purpura (ITP), polyclonal IgG antibodies against platelet surface antigens were used to induce a lethal disease. These profoundly thrombocytopenic animals were treated and cured by a single injection of IdeS.

Conclusions/significance: Novel information is provided concerning the IgG-cleaving activity of IdeS in vitro and in vivo. The highly specific and rapid elimination of IgG in vivo, the dramatic effect in a mouse model of ITP, and the lack of side effects in the treated animals, indicate that IdeS could also be used to treat IgG-driven diseases in humans.

Figure 1. IdeS cleaves IgG in human blood.

(A) Structure of IgG. The IdeS cleavage sites are indicated. (B) The following samples were separated by SDS-PAGE. Lane 1: Five µg of human polyclonal IgG in 10 µl PBS. Lane 2: Five µg of human polyclonal IgG and 1 µg of IdeS in 10 µl PBS (IgG and IdeS were preincubated for three hours at 37°C before SDS-PAGE). Lane 3: Ten µl of plasma from human blood diluted 1∶50 in PBS Lane 4: One hundred µl of human blood was preincubated with 1 µg of IdeS for three hours at 37°C. The plasma from this sample (containing approximately 20 µg/ml) was diluted 1∶50 in PBS, and 10 µl of this material was separated in lane 4. The asterisk indicates the IgG heavy chain.

Figure 2. IgG antibodies against IdeS in human sera do not interfere with the enzymatic activity.

(A) Antibody titers against IdeS in serum samples from nineteen healthy donors (D1–D19). (B) SDS-PAGE analysis of serum samples untreated (−) or preincubated with IdeS (20 µg/ml serum) for three hours at 37°C (+).

Figure 3. Invivo cleavage and removal of IgG from the blood circulation of rabbits injected with IdeS.

(A) SDS-PAGE of rabbit polyclonal IgG (5 µg in 10 µl PBS) alone (lane 1), or preincubated with IdeS (5 µg IgG and 1 µg IdeS in 10 µl PBS) for three hours at 37°C (lane 2). Bands corresponding to IdeS (61 kDa), IgG heavy chains (Hc, 56 kDa), IdeS-generated Hc fragments (31 kDa) and IgG light chains ( Lc, 25 kDa), are indicated. (B) Levels of IgG (grey bars) and IdeS (⋄) in serum samples from a rabbit injected i.v. with IdeS (5 mg diluted in 2.5 ml PBS). IgG was determined by ELISA and IdeS by Western blotting and chemoluminescence in a Chemidoc XRS Imaging system. Samples were analyzed three times and mean values±SD are indicated.

Figure 4. IdeS cures mice from lethal IgG-induced thrombocytopenia.

(A) Fourteen BALB/C mice were injected i.p. with a lethal dose of purified polyclonal rabbit IgG antibodies (1 mg IgG/ mouse, diluted in 0.25 ml PBS) raised against mouse platelets. Thirty minutes later, seven mice were treated i.p. with 0.5 mg IdeS in 0.25 ml PBS and seven with 0.25 ml PBS alone. In another experiment, twelve Balb/c mice were injected i.p. with the lethal dose of IgG. After thirty minutes, six mice were given 0.5 mg IdeS in 0.25 ml PBS, and six were given 0.25 ml PBS alone. In this case, the injections were given i.v. The thirteen IdeS treated mice (○) all survived, whereas the thirteen PBS treated mice (▪) all died within 24 hours. (B) Blood samples from the twelve mice injected with the lethal dose of purified rabbit IgG and treated i.v. with IdeS or PBS alone (six mice in each group), were analyzed using flow cytometry of the platelet population. Samples from two representative animals, one injected with IdeS and one with PBS, are shown. (C) The number of platelets in the blood samples from the twelve animals treated i.v. was counted manually; six treated with IdeS (○) and six with PBS (▪). Error bars indicate ±SEM.

Figure 5. IdeS treatment of thrombocytopenia is effective also when initiated at low platelet levels.

(A) Blood samples were taken at regular time intervals from three mice injected i.p. with a lethal dose of rabbit anti-mouse platelet IgG antibodies (1 mg IgG/mouse), and the number of platelets was determined by manual counting. (B) An additional ten mice were injected with the lethal dose of IgG. At three hours, five mice were treated i.v. with IdeS (0.5 mg in 0.25 ml PBS/mouse), while the other five received PBS alone (0.25 ml i.v/mouse). The PBS treated animals remained thrombocytopenic and died within 18–30 hours of IgG administration, whereas the IdeS treated animals all recovered their platelet levels and survived. These animals were sacrificed on day 14.