Sandra Wymann

Analysis and Functional Consequences of Increased Fab-Sialylation of Intravenous Immunoglobulin (IVIG) after Lectin Fractionation

It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab’)2 and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.

Self-reactivity in the dimeric intravenous immunoglobulin fraction.

Abstract:  Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size‐exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp‐2 cytoplasmic proteome by 2D‐PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC‐MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype–anti‐idiotype masking of auto‐specific Abs in the dimeric fraction of IVIg.

Isoagglutinin reduction by a dedicated immunoaffinity chromatography step in the manufacturing process of human immunoglobulin products

The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG‐related hemolytic events. We present in this work a modification of the manufacturing process of Privigen—a 10% l‐proline–stabilized IVIG product—that allows extensive reduction of isoagglutinin concentrations in the final product.

Donor screening reduces the isoagglutinin titer in immunoglobulin products

Hemolysis reaction is a rare class effect of therapy with intravenously administered human normal immunoglobulin (IVIG). Anti‐A/B isoagglutinins (isohemagglutinins) originating from donor plasma are considered a probable risk factor for hemolysis. We hypothesized that screening and exclusion of plasma obtained from donors with high isoagglutinin titers from the manufacturing process would produce a meaningful reduction of anti‐A/B isoagglutinin titers of the final IVIG product.

Hemolysis related to intravenous immunoglobulins is dependent on the presence of anti‐blood group A and B antibodies and individual susceptibility

Patients treated with intravenous immunoglobulins (IVIG) rarely experience symptomatic hemolysis. Although anti‐A and anti‐B isoagglutinins from the product are involved in most cases, the actual mechanisms triggering hemolysis are unclear.

Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both ‘acquired’ and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype‐antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype‐antiidiotype Ab pairs and thus in a comparable F(ab’)2 binding site availability of the different IgG fractions. Previously, we identified an increased anti‐self‐reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen‐derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen‐associated polysaccharides, classically regarded as T‐independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

Monomeric Immunoglobulin A from Plasma Inhibits Human Th17 Responses In Vitro Independent of FcαRI and DC-SIGN

Circulating immunoglobulins including immunoglobulin G (IgG) and IgM play a critical role in the immune homeostasis by modulating functions of immune cells. These functions are mediated in part by natural antibodies. However, despite being second most abundant antibody in the circulation, the immunoregulatory function of IgA is relatively unexplored. As Th17 cells are the key mediators of a variety of autoimmune, inflammatory, and allergic diseases, we investigated the ability of monomeric IgA (mIgA) isolated from pooled plasma of healthy donors to modulate human Th17 cells. We show that mIgA inhibits differentiation and amplification of human Th17 cells and the production of their effector cytokine IL-17A. mIgA also suppresses IFN-γ responses under these experimental conditions. Suppressive effect of mIgA on Th17 responses is associated with reciprocal expansion of FoxP3-positive regulatory T cells. The effect of mIgA on Th17 cells is dependent on F(ab′)2 fragments and independent of FcαRI (CD89) and DC-SIGN. Mechanistically, the modulatory effect of mIgA on Th17 cells implicates suppression of phosphorylation of signal transducer and activator of transcription 3. Furthermore, mIgA binds to CD4+ T cells and recognizes in a dose-dependent manner the receptors for cytokines (IL-6Rα and IL-1RI) that mediate Th17 responses. Our findings thus reveal novel anti-inflammatory functions of IgA and suggest potential therapeutic utility of mIgA in autoimmune and inflammatory diseases that implicate Th17 cells.