Induced Polyspecificity of Human Secretory Immunoglobulin A Antibodies: Is It Possible to Improve Their Ability to Bind Pathogens?

INTRODUCTION: As has been shown previously, various protein-modifying agents can change the antigen-binding properties of immunoglobulins. However, induced polyspecificity of human secretory immunoglobulin A (sIgA) has not been previously characterized in detail. METHODS: In the present study, human secretory immunoglobulin A (IgA) was exposed to buffers with acidic pH, to free heme, or to pro-oxidative ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens was compared using Western blotting and enzyme-linked immunosorbent assay. The ability of these agents to modulate the antigen-binding properties of human sIgA toward a wide range of pathogen peptides was investigated using an epitope microarray. RESULTS: We have shown that acidic pH, heme, and pro-oxidative ferrous ions influenced the binding of secretory IgA in opposite directions (either increasing or decreasing); however, the strongest effect was observed when using buffers with low pH. This fraction had the highest number of affected reactivities; most of them were increased and most of the new ones were toward common pathogens. CONCLUSIONS: Thus, it was shown that all investigated treatments can alter to some degree the antigen-binding of secretory IgA, but acidic pH has the most potentially beneficial effect by increasing binding to a largest number of common pathogens' antigens.

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes.

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as "genomic storm" in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous "danger" signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.

Relationship between natural and heme-mediated antibody polyreactivity.

Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies.

Mechanism and functional implications of the heme-induced binding promiscuity of IgE.

A fraction of antibodies from healthy immune repertoires binds to heme and acquires the ability to recognize multiple antigens. The mechanism and functional consequences of heme-mediated antigen binding promiscuity (polyreactivity) are not understood. Here, we used SPE7, a mouse monoclonal IgE specific for dinitrophenyl that has been thoroughly characterized at the molecular level, as a model antibody to elucidate the mechanism and functional consequences of heme-mediated polyreactivity. We first demonstrate that exposure of SPE7 to heme results in a substantial increase in its antigen binding polyreactivity. Comparison of the binding kinetics and thermodynamics of interaction of native and heme-bound SPE7 indicates that the binding of heme to SPE7 confers binding affinities in the low nanomolar range toward several antigens but has no influence on the mechanism of recognition of dinitrophenyl. In vitro cellular assays further demonstrate that heme-bound SPE7 does not promote the degranulation of basophils in the presence of new target antigens, while degranulation is observed in the presence of dinitrophenyl. Molecular docking and fluorescence spectroscopy revealed binding of heme to the variable region of SPE7 at a distance from the conventional binding site for dinitrophenyl, explaining the extension of binding polyreactivity without abrogation of the interaction with its cognate antigen. In addition, our data suggest that heme, when bound to IgE, is solvent-exposed and may serve as an interfacial cofactor mediating binding to diverse proteins. This study contributes to the understanding of the mechanisms of heme-induced antibody polyreactivity. It also paves the way toward the delineation of the functional impact of polyreactivity and cross-reactivity of IgE.

Antibody polyreactivity in health and disease: statu variabilis.

An Ab molecule or a BCR that is able to bind multiple structurally unrelated Ags is defined as polyreactive. Polyreactive Abs and BCRs constitute an important part of immune repertoires under physiological conditions and may play essential roles in immune defense and in the maintenance of immune homeostasis. In this review, we integrate and discuss different findings that reveal the indispensable role of Ag-binding polyreactivity in the immune system. First, we describe the functional and molecular characteristics of polyreactive Abs. The following part of the review concentrates on the biological roles attributed to polyreactive Abs and to polyreactive BCRs. Finally, we discuss recent studies that link Ig polyreactivity with distinct pathological conditions.

Antibody polyspecificity: what does it matter?

Polyspecificity (polyreactivity) is currently considered an intrinsic property of a subset of antibodies, primarily of naturally occurring autoantibodies. Polyspecificity is no longer viewed as a biologically irrelevant stickiness. Furthermore, the capacity to bind defined sets of unrelated antigens finds its structural explanation. What is most intriguing, the elucidation of the role of polyspecificity may promote a better understanding of specific recognition as a function of the entire immune system. The early events of immune recognition depend on polyspecific binding. Thus, the completeness of the naïve repertoires of antigen receptors is ensured. The process of immunologically-relevant antigen recognition that is initiated goes beyond simple molecular interaction with the antigenic determinants. It involves cellular cooperation and culminates in antibody response maturation. Recent findings also pave the way for the clinical application of posttranslationally induced polyspecificity.

Induced antigen-binding polyreactivity in human serum IgA.

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.

Intravenous immunoglobulins exposed to heme (heme IVIG) are more efficient than IVIG in attenuating autoimmune diabetes.

Intravenous immunoglobulins (IVIG) are known to have a therapeutic effect in some autoimmune diseases. We examined the effect of IVIG and heme-exposed IVIG on the development of immune mediated diabetes induced in C57BL/6 mice by multiple low doses of streptozotocin. IVIG were used in a dose of 200mg/kg daily for 15 days. Treatment with IVIG resulted in significant attenuation of diabetes induction as evaluated by glycemia, glycosuria and HbA1c level. Interestingly, heme-exposed IVIG had a still stronger antidiabetogenic effect. Serum levels of proinflammatory cytokines TNF-α, IFN-γ and IL17 were lower in IVIG treated animals when compared with controls, while IL10 level was higher. The number of CD4(+)Foxp3(+) cells was higher in pancreatic lymph nodes of heme-exposed IVIG treated mice. Our results show that IVIG may downregulate diabetes induction possibly by favouring induction of T regulatory cells and suggest enhanced effect upon heme-binding to IVIG.

Heterogeneous antigen recognition behavior of induced polyspecific antibodies.

Polyspecific antibodies represent a significant fraction of the antibody repertoires in healthy animals and humans. Interestingly, certain antibodies only acquire a polyspecific antigen-binding behavior after exposure to protein-modifying conditions, such as those found at inflammation sites, or used in small- and large-scale immunoglobulin purification. This phenomenon is referred to as "criptic polyspecificity". In the present study, we compare the potential of different chemical agents to induce IgG polyspecificity. Depending on the treatment used, quantitative and qualitative differences in the recognition of individual antigens from a standard panel were observed. Antibodies with cryptic polyspecificity utilized common mechanisms for the recognition of structurally unrelated antigens when exposed to a particular inductor of polyspecificity. Our study contributes to the understanding of the mechanisms underlying the cryptic polyspecificity.

Progression of lupus-like disease drives the appearance of complement-activating IgG antibodies in MRL/lpr mice.

OBJECTIVES: Nucleic acids are known to induce complement activation, which results in the masking and removal of apoptotic cells exposing nuclear components. Dysregulation of these events is characteristic of SLE, a systemic autoimmune disease characterized by the appearance of ANAs. In this study, we aimed to investigate the relationship between development of ANAs and their effect on complement activation by nucleic acids. METHODS: We used protein array technology to characterize complement activation by murine mAbs and polyclonal antibodies against various forms of nucleic acid. Serum samples from MRL/lpr mice were collected, starting before the onset of the disease till 6 months of age. Binding of IgG and its subclasses to dsDNA, ssDNA, RNA, plasmid DNA and nucleosome complexes was determined, along with C3 fixation. RESULTS: We show that complement C3 binding to various forms of nucleic acid that serve as targets in lupus is absent in normal serum. The addition of dsDNA-specific mAbs to normal serum results in the deposition of complement C3 to nucleic acids. In MRL/lpr mice, IgG antibodies against various nuclear antigens appear with ageing and disease progression. C3 binding to the antigens is somewhat delayed and suggests that accumulation or maturation of pathogenic antibodies is required for inducing C3 binding to ICs containing nucleic acids. CONCLUSIONS: C3 deposition on nuclear antigens, therefore, reflects the state of disease progression in this murine model of SLE.

Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils.

Mild modification of intravenous immunoglobulin (IVIG) has been reported to result in enhanced polyspecificity and leveraged therapeutic effects in animal models of inflammation. Here, we observed that IVIG modification by ferrous ions, heme or low pH exposure, shifted the repertoires of specificities in different directions. Ferrous ions exposed Fe(II)-IVIG, but not heme or low pH exposed IVIG, showed increased pro-apoptotic effects on neutrophil granulocytes that relied on a FAS-dependent mechanism. These effects were also observed in human neutrophils primed by inflammatory mediators or rheumatoid arthritis joint fluid in vitro, or patient neutrophils ex vivo from acute Crohn's disease. These observations indicate that IVIG-mediated effects on cells can be enhanced by IVIG modification, yet specific modification conditions may be required to target specific molecular pathways and eventually to enhance the therapeutic potential.

Intravenous immunoglobulin up-regulates the expression of the inhibitory FcgammaIIB receptor on B cells.

Intravenous immunoglobulin (IVIg) preparations are known to modulate autoimmune/inflammatory diseases through several F(ab')(2)- and Fc-dependent mechanisms. In this study, we show that the in vitro and the in vivo exposure of B lymphocytes from lupus-prone and from healthy mice to IVIg results in an increased expression of their surface inhibitory FcgammaIIB receptors. Further, this exposure enhanced the ability of a chimeric antibody, cross-linking FcgammaRIIB and immunoglobulin receptors on DNA-specific B lymphocytes, to suppress IgG anti-DNA antibody production. F(ab')(2) fragments of IVIg had a similar activity as the intact preparation, whereas Fc fragments had no effect. This study describes a novel approach with clinical relevance for modulating B lymphocyte activity.

Selective silencing of disease-associated B-lymphocytes by chimeric molecules targeting their Fc gamma IIb receptor.

The presently used approaches to silence autoreactive disease-associated B cells act indiscriminately and more specific therapies are obviously needed. In the present study, we analyze the ability of a chimeric antibody to suppress selectively pathological autoreactive B-lymphocytes in lupus-prone mice by cross-linking their surface Ig receptors with the inhibitory IgG Fc gamma RIIb receptors. The chimera was constructed by coupling an immunodominant mouse Histone 1 peptide to a rat monoclonal anti-mouse CD32 (Fc gamma RIIb) antibody. The administration of these chimeric molecules to MRL/lpr mice with initial and with full-blown disease resulted in the reduction of the levels of IgG anti-Histone 1 antibodies, of the albuminuria levels, of the size of lymphoid organs and in prevention of the development of skin lesions. The observed effect was limited to lupus-associated B cells only, as the treatment did not decrease the IgG antibody response to an administered foreign antigen. This study demonstrates the possibility to silence selectively autoreactive B cells and to delay the progression of an autoimmune disease using chimeric antibody molecules.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries.

Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients' sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

Insight into the mechanism of the acquired antibody auto-reactivity.

A fraction of antibodies present in all healthy individuals begins to recognize large number of self-antigens only after a transient exposure to certain protein-destabilizing conditions, including low or high pH, high salt concentration, chaotropic factors and redox-active agents. Recent findings on the molecular mechanisms of the inducible antibody auto-reactivity, obtained by using kinetic and thermodynamic analysis on the interactions of single monoclonal antibody with its cognate antigen are discussed in the review.

Functional variability of antibodies upon oxidative processes.

Reactive oxygen species (ROS) released from activated phagocytes are involved in the innate immune defense against pathogens. However, when released in excess and when the antioxidant systems are impaired, ROS may induce cellular and tissue damage and dissociation of iron ions or iron containing compounds (heme) from protein-bound state. Free iron ions and free heme are prooxidative. Immunoglobulins usually perform their biological functions at sites of inflammation, where they may encounter reactive oxygen species and/or redox active compounds. It has been demonstrated that the exposure of some antibodies to heme, to transition metal ions or to reactive oxygen species induces an appearance of new binding specificities for various autoantigens. This review article is devoted to the interplay between redox active agents and antibodies. The biological significance of the appearance of new antigen binding specificities on antibodies after exposure to redox-active agents is discussed.

An antibody-based construct carrying DNA-mimotope and targeting CR1(CD35) selectively suppresses human autoreactive B-lymphocytes.

There is an urgent and unmet need for therapeutic agents targeting selectively disease-associated B-lymphocytes in autoantibody-mediated diseases. We have constructed a chimeric molecule able to cross-link cell surface immunoglobulin with the inhibitory complement receptor type 1 (CD35) on DNA-specific B cells from SLE (systemic lupus erythematosus) patients with the aim of selectively silencing them. This engineered molecule is made of copies of the DNA-mimotope peptide DWEYSVWLSN coupled to a monoclonal anti-CD35 antibody. We found that the DNA-like peptide chimera induced a dose-dependent decrease in the number of IgG anti-dsDNA antibody producing cells when PBMCs of lupus patients were cultured in its presence. Our data present evidence that clustering BCR and the inhibitory CR1 on disease-associated autoreactive B-lymphocytes selectively suppresses autoantibody production.

Transition towards antigen-binding promiscuity of a monospecific antibody.

Polyspecificity is defined as the ability of a given antibody molecule to bind a large panel of structurally diverse antigens. A fraction of circulating IgG in all healthy individuals acquires promiscuous antigen-binding activity only after a transient exposure to certain protein destabilizing factors. The molecular mechanisms of this phenomenon are not well understood. Exposures to protein destabilizing agents are common steps in immunoglobulin isolation and purification processes. We performed kinetic and thermodynamic analyses using surface plasmon resonance-based technique in order to characterize the interactions of a single mouse monoclonal antibody to its cognate antigen before and after induction of promiscuous antigen-binding activity. The obtained results, suggest that enhanced antigen binding activity induced by exposure to mild denaturing condition resulted from an increase in the structural flexibility of the antigen-binding site. Further pH and ionic strength-dependence analyses of the antibody/antigen interactions demonstrated that the transition to promiscuous antigen-binding was accompanied by a change in the type of non-covalent forces involved in the complex formation. Moreover, from this study, it is evident that an antibody molecule could use two distinct thermodynamic pathways for binding to the same antigen while retaining the same value of the binding affinity. The obtained results may contribute to the understanding of the molecular mechanisms that lay behind natural antibody polyspecificity.