The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe

Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus, are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens.

The mycoplasma surface proteins MIB and MIP promote the dissociation of the antibody-antigen interaction

Mycoplasma immunoglobulin binding (MIB) and mycoplasma immunoglobulin protease (MIP) are surface proteins found in the majority of mycoplasma species, acting sequentially to capture antibodies and cleave off their VH domains. Cryo–electron microscopy structures show how MIB and MIP bind to a Fab fragment in a “hug of death” mechanism. As a result, the orientation of the VL and VH domains is twisted out of alignment, disrupting the antigen binding site. We also show that MIB-MIP has the ability to promote the dissociation of the antibody-antigen complex. This system is functional in cells and protects mycoplasmas from antibody-mediated agglutination. These results highlight the key role of the MIB-MIP system in immunity evasion by mycoplasmas through an unprecedented mechanism, and open exciting perspectives to use these proteins as potential tools in the antibody field.

A Protein A based Staphylococcus aureus vaccine with improved safety

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses. One such factor, Staphylococcal protein A (SpA) encompasses five Immunoglobulin binding domains (IgBDs) that associate with the Fcγ domain to block phagocytosis. IgBDs also associate with the Fab domain of VH3-idiotypic IgM which activates B cells with the resulting secretion of antibodies that cannot bind determinants of S. aureus. SpA crosslinking of VH3-idiotypic IgG and IgE receptors of mast cells and basophils promotes histamine release and anaphylaxis. Previous work demonstrated the safety, immunogenicity, and protective efficacy of SpAKKAA, a variant partially defective for VH3-idiotypic Ig cross-linking, in murine models of S. aureus. Compared to mice (10%), humans produce significantly more VH3-idiotypic B cells (50%), prompting a search for safer SpA variants that may be suitably developed as clinical-grade vaccines for efficacy testing in humans. Here, we report the identification of such variants.

ACE2 Is a Prognostic Biomarker and Associated with Immune Infiltration in Kidney Renal Clear Cell Carcinoma: Implication for COVID-19

Background. KIRC is one of the most common cancers with a poor prognosis. ACE2 was involved in tumor angiogenesis and progression in many malignancies. The role of ACE2 in KIRC is still ambiguous. Methods. Various bioinformatics analysis tools were investigated to evaluate the prognostic value of ACE2 and its association with immune infiltration in KIRC. Results. ACE2 was shown to be downregulated in KIRC at the mRNA and protein level. Low expression of ACE2 protein in KIRC patients was observed in subgroup analyses based on gender, age, weight, tumor grade, and cancer stage. Upregulation of ACE2 in KIRC was associated with a favorable prognosis. ACE2 mRNA expression showed a positive correlation with the abundance of immune cells (B cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells) and the level of immune markers of different immune cells in KIRC. ACE2 expression could affect, in part, the immune infiltration and the advanced cancer stage. Moreover, enrichment analysis revealed that ACE2 in KIRC were mainly involved in translation factor activity, immunoglobulin binding, metabolic pathways, transcriptional misregulation in cancerous cells, cell cycle, and ribosomal activity. Several ACE2-associated kinases, miRNA, and transcription factor targets in KIRC were also identified. Conclusion. ACE2 was downregulated in KIRC and served as a prognostic biomarker. It was also shown to be associated with immune infiltration.

A proteomics-based method for identifying antigens within immune complexes

A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

Universal enzyme-linked immunosorbent assays (ELISA) and utility in the immunodiagnosis of Salmonellosis

The aims of this research are confirming the feasibility of hybrid immunoglobulin-binding reagents, its used in ELISAs for IgG/IgY detection and detecting specific antibodies against an infectious microorganism (Salmonella spp) in various animal species using a universal diagnostic ELISA. Hybrid immunoglobulin-binding bacterial proteins (IBP) including recombinant protein LA, recombinant protein LG, and recombinant protein AG have been produced for improvement of their binding affinity to a much larger number of immunoglobulins. This hybrid bacterial protein thus represents a powerful tool for binding, detection, and purification of immunoglobulins and their fragments. However, SpLA-LG-peroxidase and SpLAG-anti-IgY-peroxidase were produced by the periodate method. They have shown to be effective reagents. Their binding affinity to immunoglobulins surpasses previous hybrid IgG-binding proteins reported, including the most known SpAG, SpLA and SpLG. The IgY fraction was isolated from the egg yolks of a variety of birds including species of chicken, bantam hen, guinea hen, quail, goose, duck, wild and domestic pigeon, parakeet, cattle egret, pheasant, and ostrich. The IgY fraction was isolated by the chloroform-polyethylene glycol (PEG) method. An ELISA for anti-Salmonella spp antibodies was employed with some modifications to determine the presence of antibodies in humans, laying hens, geese, quails, and pigeons. Salmonella are motile, flagellated rod-shaped zoonotic pathogens which may survive with or without oxygen. They belong to the family Enterobacteriaceae and is implicated with typhoid fever and food-borne illnesses. This pathogen is associated with several diseases, which may become fatal and negatively impact the health of individuals and various economies globally. The poultry industry is most impacted and vulnerable to the onslaught of this pernicious microbe.

Use of immunoglobulin-binding bacterial proteins in immunodetection

One of the aim of this study was to make universal chimeric conjugates to react with both avian and mammalian immunoglobulins in enzyme-linked immunosorbent assays (ELISAs). The periodate method was used in the conjugation process of cross-linking horseradish peroxidase to immunoglobulin-binding proteins (IBP) including staphylococcal protein A (SpA), streptococcal protein G (SpG) and peptostreptococcal protein L (SpL). By mixing up these three conjugates another four hybrid protein conjugates were created including protein LA (SpLA), protein LG (SpLG), protein AG (SpAG) and protein LAG (PLAG). Thirty-five ELISAs were standardized by a probabilistic combination of these immunoreagents. By using a panel of mainly mammalian immunoglobulins their reproducibility was checked by the determination of coefficient of variations (CV) for each one of the IgG-IBP binding. The source of immunoglobulins was their purification by affinity chromatography using a commercially available kit (PURE-1A). The other aim was to immunize chicken with the peptide fragment 254-274 of gp120 to produce anti-HIV peptide hyper-immune egg. Cats and rats were fed these eggs for a determined period until they produced the anti-HIV peptide antibody, which was tested by an indirect SpLA-ELISA and dot blot analysis that corroborated the production of anti-HIV antibodies by the mammalian species including positive humans samples for HIV. We conclude that the single and hybrid immunoglobulin-binding protein were effective in their binding capacity to immunoglobulins from a variety of mammalian species. The potential use of this proteins is in the arena of immunodiagnosis and immunoglobulin detection. Dot blot analysis proves effective in the detection of HIV anti-gp120 antibodies in several animal species. These antibodies can be used as reagents in the development of immunodiagnostic tests or experimental vaccines.