Decreased protective efficacy of reduced and alkylated human immune serum globulin in experimental infection with Haemophilus influenzae type b

Abstract

Conventionally prepared immune serum globulin frequently produces severe side effects when administered intravenously. A modified preparation in which 4 to 5 interchain disulfide bonds have been reduced and alkylated has been made for intravenous use. However, reduction and alkylation may affect Fc-mediated functions of immunoglobulin G, particularly its ability to fix complement by the classical pathway. To determine whether reduction and alkylation alters the protective activity of immune serum globulin in vivo we compared it with two less harshly prepared globulins (pH 4 treated or ultrafiltered) in an infant rat model of Haemophilus influenzae b infection. Antibody binding to the capsular and noncapsular components of H. influenzae b and in vitro bactericidal activity were similar in the globulin preparations. Infant rats were treated with various doses of globulins adjusted to provide identical concentrations of anticapsular antibodies as measured by the Farr radioactive antigen binding assay. At high doses of anticapsular antibody (>1,500 ng per pup), all preparations protected well. At marginal doses (750 ng per pup), however, rats given reduced and alkylated globulin had a significantly greater incidence of bacteremia (P < 0.05), meningitis (P < 0.01), and death (P < 0.05) and a higher magnitude of bacteremia (P < 0.02) than rats who received pH4-treated or ultrafiltered globulins. These differences were not due to differences in anticapsular antibody concentrations achieved in the serum. The 50% protective serum concentrations of anticapsular antibody in this model were 200 to 300 ng/ml for reduced and alkylated globulin and 100 to 200 ng/ml for acid-treated globulin. Absorption of the globulins with purified H. influenzae b capsule reduced in vitro bactericidal activity and rat protective activity. However, the magnitude of bacteremia was lower in rats receiving absorbed pH 4-treated globulin than in those receiving absorbed reduced and alkylated globulin (P < 0.05). We conclude that reduced and alkylated immunoglobulin G provides significantly less protective activity against H. influenzae b infection in this model than globulins not so modified, and we suggest that the altered Fc function of the immunoglobulin G, such as the decreased ability to fix complement by the classical pathway or decreased Fc-mediated opsonization, may be responsible for this impairment.

Effect of complement depletion on anticapsular-antibody-mediated immunity to experimental infection with Haemophilus influenzae type b

“…Infant rats that received single-donor human IgG or IgM Hib CP antibody in doses giving serum antibody levels of 200 to 2,000 ng/ml were protected from Hib bacteremia, meningitis, and death (Table 1), consistent with previous findings (16)(17)(18)(19). Administration of CoVF, however, abrogated the protective effects of both IgG and IgM Hib CP antibody even when levels in serum were many times the minimum protective level.…”

“…A streptomycin-resistant mutant of Hib Eagan (E-1) was freshly subcultured for each experiment and used in the animal experiments. Inocula for animal challenge were prepared as previously described (18,19), and cultures of tail blood and spinal fluid of rats were grown on supplemented brain heart infusion agar containing 40 ,ug of streptomycin per ml and 5 U of bacitracin per ml to inhibit growth of contaminants.…”

“…After 18 h, 30 to 50 RI of blood was obtained by tail vein puncture for assay of antibody to capsule or determination of complement levels via CH50 or both. When 6 days old, animals were challenged intraperitoneally with 5 x 103 Hib, and blood bacterial density was monitored 1, 2, and 4 days after Hib inoculation (18,19). Cultures of cerebrospinal fluid were obtained 2 days after Hib challenge by holding each pup with head flexed and using a 27-gauge needle to puncture the cisterna magna (18).…”

Relative functional activity of purified human immunoglobulin G against a type III group B streptococcal strain

“…Hetherington and Giebink reported that the opsonic activity of IgG prepared by chromatography with DEAE-Sephadex is greater than that of reduced and alkylated IgG against Staphylococcus aureus and Streptococcus pneumoniae (2). Schreiber et al also reported that pH 4-treated IgG is more protective in infant rats against Haemophilus influenzae type b than reduced and alkylated IgG (8). In this study, IgG separated from whole serum by QAE-Sephadex appeared to be even more protective in vivo against the type III GBS than IgG prepared from Cohn fraction II that had been reduced and alkylated or treated at pH 4.…”

Effect of immunoglobulin G (IgG) interchain disulfide bond cleavage on efficacy of intravenous immunoglobulin for immune thrombocytopenic purpura (ITP)

“…S-sulfonation [13] or S-alkylation following reduction [14] under mild conditions cleaves the interchain, but not the intrachain, disulfide bonds of IgG, resulting in decreased activity of some effector functions, such as complement activation. Recently, Dall'Acqua et al [15] showed that recombinant IgG molecules without inter-H chain disulfide bonds had decreased antibody-dependent cellular cytotoxicity.…”