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One of the major reasons behind the limited success of vaccine candidates against all forms of leishmaniasis is the inability of parasitic antigens to induce robust cell-mediated immunity and immunological memory. Here we find, for the first time, that the adjuvantation of whole-killed Leishmania vaccine (Leishvacc) with anti-CD200 and anti-CD300a antibodies enhances CD4+ T cells mediated immunity in vaccinated mice and provides protection against wild-type parasites. The antibody adjuvantation, either alone or with a TLR4 agonist monophosphoryl A (MPL-A), induced the production of pro-inflammatory cytokines viz., IFN-γ, TNF-α, and IL-2 by antigen experienced CD4+ T cells, and also enhanced their rate of conversion into their memory phenotypes against Leishvacc antigens. The antibody adjuvanted vaccine also promoted the generation of IgG2a-mediated protective humoral immunity in vaccinated mice. Further, the mice vaccinated with antibodies adjuvanted vaccine showed strong resilience against metacyclic forms of L. donovani parasites as we observed reduced clinical features such as splenomegaly, hepatomegaly, granulomatous tissues in the liver, and parasitic load in their spleen. The findings of this study demonstrate that the anti-CD200 and anti-CD300a antibodies have potential to increase the protective efficacy of the whole-killed Leishmania vaccine, and opens up a new gateway to diversify the roles of immune checkpoints in vaccine development against leishmaniasis. • Anti-CD200/CD300a antibodies adjuvantation enhances vaccine antigen immunogenicity. • Antibodies (Abs) adjuvanted Leishvacc enhances dendritic and CD4 T cells functions. • Abs adjuvanted Leishvacc provides better protection against wild-type parasites. [ABSTRACT FROM AUTHOR]

The essence of the growth and development of therapeutic conventional monoclonal antibodies (MAbs) for the treatment of various disorders is the aptitude of MAbs to precisely bind a target antigen and neutralise or promote its activity. However, the conventional antibodies are monoclonal i.e., both paratopes bind to the same epitope. But most of the pathophysiological conditions are multifaceted, hence targeting/blocking/inhibition of more than one epitope/antigen is more promising than one epitope/antigen. Polyspecific antibodies (PsAbs) have the potential to concurrently bind to more than one target and are the next-generation antibodies that augment efficacy in both clinical and non-clinical contexts. Thus, the trend of engineering and developing various formats of PsAbs is emerging. In this review, we have briefly discussed the importance of antibody polyspecificity and PsAbs approved for clinical use. Subsequently, we have discussed the role of TNF-α and IL-23 in inflammatory diseases and stressed the need for developing anti-TNF-α and anti-IL-23 bispecific antibodies. • Monoclonal antibodies targeting single epitope/antigen having number of promising clinical and non-clinical applications. • Now the trend is changing to polyspecific antibodies targeting more than one epitope/antigen simultaneously. • Chronic inflammation claims 3 deaths out of 5 individuals worldwide. • TNF-α and IL-23 both are main drivers of chronic inflammation, inhibiting their interactions with their respective receptors using polyspecific antibodies is one of the intriguing treatments to control chronic inflammation. One promising approach to reducing inflammation is the use of PsAb to block TNFα and IL-23 from binding to their receptors. [ABSTRACT FROM AUTHOR]

HEMGN belongs to the Cancer/testis antigens (CTAs), which are expressed in various types of human cancers and have received particular attention in cancer immunotherapy. However, the potential function of HEMGN involved in lung cancer and the immune response is not yet elucidated. HEMGN expression in lung adenocarcinoma (LUAD) was estimated via the Tumor Immune Estimation Resource (TIMER), The Cancer Genome Atlas (TCGA), The University of Alabama at Birmingham Cancer data analysis Portal (UALCAN), and Human Protein Atlas databases. The prognostic role of HEMGN was investigated by Gene Expression Profiling Interactive Analysis (GEPIA), PrognoScan, and Kaplan-Meier plotter databases. The associations between HEMGN and clinicopathological parameters were analyzed with UALCAN database. Then, immunohistochemical and Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) analysis were performed to further verify the associations in tissue or serum samples. Serum from patients were detected for HEMGN antibody by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to detect immune cell infiltration in peripheral blood of patients with LUAD. In addition, Gene Set Enrichment Analysis (GSEA) was conducted to investigate the functional role of HEMGN. Furthermore, we obtained the somatic mutation data from the TCGA LUAD dataset and analyzed the mutation profiles with "maftools" package. Finally, we evaluated the associations between HEMGN and immune infiltration level and the characteristic markers of immune cells in TIMER, GEPIA, and CIBERSORT. The mRNA and protein expressions of HEMGN were significantly decreased in LUAD patients. High HEMGN expression was remarkably associated with better prognosis in LUAD patients. The concentration levels of anti-HEMGN antibody in LUAD were significantly higher than that in healthy individuals and were closely correlated with clinical stage. In addition, HEMGN was involved in distinct typical genomic alterations in LUAD. GSEA demonstrated that HEMGN was significantly connected with immunity and substance metabolism. Notably, HEMGN was significantly related to immune infiltrates, including B cells, CD8 + T cells, CD4 + T cells, neutrophils, macrophages, dendritic cells (DCs), and various kinds of functional T cells. Furthermore, HEMGN had a significant association with diverse immune gene markers. HEMGN can be considered as a prognostic biomarker of LUAD and is associated with immune infiltration. • The mRNA and protein expressions of HEMGN were significantly decreased in LUAD. • High HEMGN expression was remarkably associated with better prognosis in LUAD patients. • Cancer/testis antigen HEMGN is significantly associated with immune response and inflammatory activities, as well as immune cell infiltration and diverse immune marker sets. [ABSTRACT FROM AUTHOR]

Allelic variants of HLA-DRB1 have been associated with a variety of autoimmune and infectious diseases. Although the precise molecular mechanisms by which HLA-DRB1 alleles predispose to a particular disease are currently unclear, it has been shown that mRNA expression levels of HLA-DRB1 are dependent on the different alleles. We aimed to measure HLA-DR beta chain levels in peripheral blood mononuclear cells of individuals carrying HLA-DRB1 *03:01/*04:01 and HLA-DRB1 *03:01/*15:01 alleles by western blotting, using five commercially-available HLA-DRB antibodies. We observed highly heterogeneous binding of the tested antibodies to the different allelic forms of the HLA-DR beta chain. Overall, we show that current immunological research that employs available antibodies to detect HLA-DR beta chains is biased towards detection of specific variants of the protein; this may cause significant discrepancy in quantification of protein expression in a heterogeneous human population. • RNA expression levels of HLA-DRB1 are dependent on the different HLA-DRB1 alleles. • Tested commercial HLA-DRB antibodies detect particular allelic forms of HLA-DRB1. • Novel tools are needed to study allele-specific protein expression of HLA-DRB1. [ABSTRACT FROM AUTHOR]

Human leukocyte antigen (HLA)-A2 antibody contributes to the pathogenesis of transfusion-related acute lung injury (TRALI) via polymorphonuclear neutrophil (PMN) activation, but the signaling pathways involved this process remain largely undefined. In this study, we sought to study the signaling pathways involved in the pathogenesis of HLA-A2-induced TRALI. Lipopolysaccharide (LPS), and the plasma from the HLA-A2 antibody-positive donors were utilized to establish a rat model of TRALI. Human pulmonary endothelial cells (HPMECs) were in vitro co-cultured with HLA-A2 antibody-treated PMNs and then treated with LPS to induce a cytotoxicity model. The effects of HLA-A2 antibody on HPMEC injury were evaluated in this model. Besides, dasatinib was used to block the Src phosphorylation to explore whether Src involved in the TRALI or HPMEC injury induced by HLA-A2 antibody. The HLA-A2 antibody plus LPS induced TRALI and stimulated PMN activation in rats. HLA-A2 antibody-induced TRALI could be attenuated via depletion of PMN. HLA-A2 antibody activated NF-κB and NLRP3 inflammasome. In addition, HLA-A2 antibody aggravated the HPMEC injuries and the release of PMN surfaces makers, but dasatinib treatment reversed this effect, indicating that HLA-A2 antibody activated PMNs and exacerbated TRALI by stimulating phosphorylation of Src followed by activation of NF-κB and NLRP3 inflammasome, which was validated in vivo. In summary, HLA-A2 induced PMNs by activating NF-κB/NLRP3 inflammasome via phosphorylated-Src elevation, thereby exacerbating TRALI. This study highlights promising target for the treatment of antibody-mediated TRALI. • HLA-A2 antibody activates phosphorylated Src in PMNs. • HLA-A2 antibody stimulates the release of PMN surface adhesion factors. • HLA-A2 antibody activates NF-κB/NLRP3 inflammasome via Src phosphorylation. • HLA-A2 antibody exacerbates TRALI by activating phosphorylated Src in PMNs. • This study provides new therapeutic target for TRALI. [ABSTRACT FROM AUTHOR]

• Spearman's ρ correlation is used for understanding anti-HLA cross-reactivities. • Spearman's ρ clearly identifies serological subgroups. • The dispersion of the Spearman ρ values is explained by an eplet approach. • The Spearman's ρ values are linked to the number of matched eplets. Identification of anti-human leukocyte antigen (HLA) antibodies (Abs) is based on Luminex™ technology. We used bioinformatics to (i) study the correlations of mean fluorescence intensities (MFIs) for all the possible allele pairs, and (ii) determine the degree of epitope homology between HLA antigens. Using MFI data on anti-HLA Abs from 6000 Luminex™ assays, we provide an updated overview of class I and II HLA antigen cross-reactivity in which each node corresponded to an allele and each link corresponded to a strong correlation between two alleles (Spearman's ρ > 0.8). We compared these correlations with the serological groups and the results of an epitope analysis. The strongest correlations concerned allele-specific Abs directed against the same antigen. For the HLA-A locus, the highest values of Spearman's ρ reflected broad specificity. For the HLA-B locus, graphs defined the HLA-Bw4 public epitope, and correlations between HLA-A and -B alleles were only present for beads with the same Bw4 public epitope. For the HLA-C locus, we identified two groups that differed with regard to their KIR ligand subclassification. Lastly, the HLA-DRB1 subgroups were part of a network. In the epitope analysis, Spearman's ρ was related to the number of matched epitopes within pairs of alleles. The combination of Spearman's ρ with simple, undirected graphing constitutes an effective tool for understanding routinely encountered cross-reactivity profiles. Based on this model, we have implemented an online data visualization tool available at http://cusureau.pythonanywhere.com/. [ABSTRACT FROM AUTHOR]

• Two recombinant truncated PKM2 (M-tPKM2, R-tPKM2) were designed via in silico analysis. • M-tPKM2 had one epitope & similar structure to PKM2, R-tPKM2 had various epitopes & different structure. • There was no significant difference between antibodies reaction with their antigens. • Ability of antisera raised against M-tPKM2 to detect the PKM2 were significantly higher. • Monoclonal antibody against M-tPKM2 could recognized PKM2 in MCF7 cells lysate. The main challenge in the development of antibody is to select the appropriate antigen particularly when a truncated protein is used for immunization or as vaccine antigen. In previous studies, fragment selection was mainly based on epitopes and less often on the structure. Fewer studies have paid attention to the prediction of the truncated protein 3D structure and retained its similarity in the native and truncated proteins. Here we used in silico analysis to select two fragments of Pyruvate Kinase M2 (PKM2), as a tumor marker. One fragment, M-tPKM2, had a shorter sequence with one epitope although the predicted 3D structure was similar to the native PKM2. The other fragment, R-tPKM2, had a longer sequence and thus more epitopes, but had a different structure from the native PKM2. Recombinant truncated proteins were expressed in E. coli and purified via affinity chromatography. Secondary structure elements in purified proteins were determined by Circular Dichroism, then they were utilized to develop antibodies in mice. Both antigens could elicit high immune response against themselves (OD 450 = 3.326 ± 0.562 for M-tPKM2; OD 450 = 3.562 ± 0.110 for R-tPKM2). However, significantly higher response against PKM2 was observed among the mice immunized with M-tPKM2 (p < 0.0001 by One way ANOVA followed by Tukey's post hoc comparison). Also, the monoclonal antibody produced against the M-tPKM2 could recognize the native PKM2 in the MCF7 cells. Our finding suggested that for the purpose of designing an antigen with the ability to produce a potent antibody against the target protein, it is better to select sequences which have a similar structure in truncated and native proteins, even at the cost of having shorter sequences and fewer epitopes. [ABSTRACT FROM AUTHOR]

Highlights • Antibodies are an essential mechanism of protection against infectious disease. • Antibody responses start when a B cell acquires antigen, often from the surfaces of another cells. • B cell antigen acquisition is regulated by immune synapse mechanics. • Quantifying immune synapse mechanics is now possible using physical manipulation and nanoscopic mechanosensors.. • Understanding B cell mechanosensitivity will open new avenues for particle-based vaccine design. Abstract B cell encounter with antigen displayed on antigen-presenting cells leads to B cell immune synapse formation, internalisation of the antigen, and stimulation of antibody responses. The sensitivity with which B cells detect antigen, and the quality and quantity of antigen that B cells acquire, depend upon mechanical properties of the immune synapse including interfacial tension, the strength of intermolecular bonds, and the compliance of the molecules and membranes that participate in antigen presentation. In this review, we discuss our current understanding of how these various physical parameters influence B cell antigen extraction in the immune synapse and how a more comprehensive understanding of B cell mechanics may promote the development of new approaches to stimulate the production of desired antibodies. [ABSTRACT FROM AUTHOR]

Nanobodies represent the next-generation antibody-derived biologics with significant advances over conventional antibodies. Several rapid and robust techniques for isolating highly specific nanobodies have been developed. Antigen specific nanobodies are selected from constructed nanobody libraries, which can be classified into 3 main types: immune library, naïve library, and semisynthetic/synthetic library. The immune library is the most widely used strategy for nanobody screening. Target specific nanobodies are highly enriched in immune libraries than in non-immune libraries; however, it is largely limited by the natural antigenicity of antigens. The naïve library is thus developed. Despite the lack of somatic maturation, protein engineering can be employed to significantly increase the affinities of selected binders. However, a substantial amount of blood samples collected from a large number of individual animals is a prerequisite to ensure the diversity of the naïve library. With this issue considered, the semisynthetic/synthetic library may be a promising path toward obtaining a limitless source of nanobodies against a variety of antigens without the need of animals. In this review, we summarize the state-of-the-art screening technologies with different libraries. The approaches presented here can further boost the diverse applications of nanobodies in biomedicine and biotechnology. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) is associated with multiple sclerosis (MS), and antibodies to the EBV nuclear antigen-1 (EBNA-1) are consistently increased in MS patients. The hypothesis of this study is that anti-EBNA-1 antibodies cross-react with a self antigen in MS patients. We affinity purified anti-EBNA-1 antibodies from human plasma, used the anti-EBNA-1 to immunoprecipitate antigens from human brain, and identified bound antigens with mass spectrometry. Anti-EBNA-1 consistently bound heterogeneous nuclear ribonucleoprotein L (HNRNPL). We expressed both the long and short isoforms of this protein, and verified with Western blots and ELISA that the long isoform cross-reacts with EBNA-1. Immunohistochemistry demonstrated that anti-EBNA-1 bound to an antigen in the nucleus of cultured rat central nervous system cells. ELISA demonstrated the presence of antibodies to HNRNPL in the plasma of both healthy controls and MS patients, but anti-HNRNPL was not increased in MS patients. We conclude that HNRNPL is an autoantigen which cross-reacts with EBNA-1. The relevance of this autoantigen to MS and other autoimmune diseases remains to be investigated. [ABSTRACT FROM AUTHOR]

Autoimmune destruction of pancreatic islets in the nonobese diabetic (NOD) mouse is driven by T cells recognizing various autoantigens mostly in insulin-producing beta-cells. To investigate if T-cell accumulation in islets during early insulitis is clonally predetermined, we compared the complementarity determining regions (CDR3) of T-cell receptor (TCR)β-chains present in islet-infiltrating T cells in young prediabetic NOD mice. High-throughput sequencing of TCRβ-chain DNA extracted from islets of 7-wk old NOD mice revealed a biased TCRβ-chain repertoire in all mice, as a restricted number of clones (17–36 clones) was highly overrepresented and made over 20% of total islet repertoire in each mouse. Among these clones, various Vβ and Jβ families were present but certain VβJβ combinations such as TRBV19-0-TRBJ2-7 and TRBV13-3-TRBJ2-5 were highly shared between individual mice. On TCRβ-chain CDR sequence level, many islet clones (72–146) were shared between at least two individual mice. None of them was among expanded clones in both, suggesting considerable stochasticity in the interactions between TCR and peptide-MHC, even with a limited range of autoantigens. A comparison of islet-CDR3-sequences with CRD-sequences from other tissues revealed clonal overlap with pancreatic lymph node and gut, but these repertoires did not overlap together. Our results suggest that antigen-specific T cells are expanded in pancreatic lymph node and islets, but different specificities expand in individual mice. Some islet-infiltrating T-cell specificities may have a distinct origin shared with gut-infiltrating T cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] Heme induces polyreactivity of IgG cloned from splenic marginal zone B-cell lymphoma. [•] Heme-induced polyreactive antibody binds various autoantigens. [•] Induction of polyreactivity does not affect Fc-fragment dependant binding of IgG. [•] Heme-induced polyreactivity of BCR might have repercussions for the pathology. [ABSTRACT FROM AUTHOR]

Abstract: The tumor antigen NY-ESO-1 is one of the most antigenic cancer-testis antigens, first identified by serologic analysis of a recombinant cDNA expression library (SEREX). NY-ESO-1 is expressed in different types of cancers including melanoma. NY-ESO-1-specific spontaneous humoral and cellular immune responses are detected in a large proportion of patients with advanced NY-ESO-1-expressing cancers. Therefore NY-ESO-1 is a good candidate antigen for immunotherapy. Although cellular immune responses to NY-ESO-1 are well characterized, much less is known about the humoral immune responses. In this study, we finely mapped linear antibody epitopes using sera from melanoma patients and shorter overlapping peptide sets. We have shown that melanoma patients’ humoral immune systems responded to NY-ESO-1 differently in each individual with widely differing antibody specificity, intensity and antibody subtypes. This knowledge will help us further understand anti-tumor immunity and may also help us to monitor cancer progress and cancer vaccine efficacy in the future. [Copyright &y& Elsevier]

Abstract: Injection of antigens coupled to antibodies against the dendritic cell (DC) surface molecule Clec9A has been shown to produce strongly enhanced antibody responses even without co-administration of adjuvants, via antigen presentation by DC on MHC class II and consequent production of follicular helper T cells. A series of mutant mice were tested to determine the DC subtypes responsible for this MHC II presentation of targeted antigen, compared to presentation of antigen on MHC I. A new clec9A null mouse was developed; these mice did not give enhanced antibody production, confirming the response was dependent on Clec9A-expressing DC. However targeting of antigen to Clec9A in batf3 null mice produced enhanced antibody responses despite the marked reduction in CD8+ DC, the major Clec9A-expressing DC subtype. This was shown to be dependent on efficient MHC II presentation by minor Clec9A-expressing DC subtypes in the environment of the Batf3−/− mice, namely early cells of the CD8 DC lineage and the plasmacytoid-related CD8+ DC subset, but not by plasmacytoid cells themselves. However in normal mice most MHC II presentation of the Clec9A-targeted antigen was by the major CD8+ DC population, the DC also responsible for presentation on MHC I. [Copyright &y& Elsevier]

Abstract: The current model of antigen assembly with major histocompatibility complex (MHC) class I molecules posits that interactions between the tapasin N-terminal immunoglobulin (Ig)-like domain and the MHC class I peptide-binding groove permit tapasin to regulate antigen selection. Much less is known regarding interactions that might involve the tapasin C-terminal Ig-like domain. Additionally, the tapasin transmembrane/cytoplasmic region enables tapasin to bridge the MHC class I molecule to the transporter associated with antigen processing (TAP). In this investigation, we made use of two tapasin mutants to determine the relative contribution of the tapasin C-terminal Ig-like domain and the tapasin transmembrane/cytoplasmic region to the assembly of MHC class I molecules. Deletion of a loop within the tapasin C-terminal Ig-like domain (Δ334–342) prevented tapasin association with the MHC class I molecule Kd. Although tapasin Δ334–342 did not increase the efficiency of Kd folding, Kd surface expression was enhanced on cells expressing this mutant relative to tapasin-deficient cells. In contrast to tapasin Δ334–342, a soluble tapasin mutant lacking the transmembrane/cytoplasmic region retained the ability to bind to Kd molecules, but did not facilitate Kd surface expression. Furthermore, when soluble tapasin and tapasin Δ334–342 were co-expressed, soluble tapasin had a dominant negative effect on the folding and surface expression of not only Kd, but also Db and Kb. In addition, our molecular modeling of the MHC class I–tapasin interface revealed novel potential interactions involving tapasin residues 334–342. Together, these findings demonstrate that the tapasin C-terminal and transmembrane/cytoplasmic regions are critical to tapasin''s capacity to associate effectively with the MHC class I molecule. [Copyright &y& Elsevier]

Abstract: The polymeric immunoglobulin receptor (pIgR) plays a pivotal role in mucosal immune protection by transporting secretory immunoglobulins to mucosal epithelia, and protecting them from proteolytic degradation. It has been reported that a homolog of the pIgR has a similar role in teleost fish. Considering the role pIgR has in mucosal defenses, this study was initiated to characterize a possible pIgR homolog in Atlantic salmon (Salmo salar) and its relatedness to pIgR of other vertebrates and similar molecules. Two pIgR-like cDNAs and genes of Atlantic salmon (Salsal pIgR and Salsal pIgRL) were cloned and analyzed. In addition, we gathered sequence information of CMRF35-like molecules (CLM) 1, 7, and 8 (designated as CD300 in humans) and made a comparative evaluation to that of the Salsal pIgR and Salsal pIgRL polypeptides. Salsal pIgR and Salsal pIgRL, like pIgR in other teleosts, are composed of two IG V domains, a connecting, a transmembrane, and a cytoplasmic region. The same holds true for Atlantic salmon CLM1 and CLM7, except that they possess putative immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic tails. The abundance of Salsal pIgR transcript is significantly higher than Salsal pIgRL and CLM in the skin, while Salsal pIgRL transcripts were abundant in the gills, depicting their possible tissue-specific role in mucosal immunity. To further highlight the roles of these molecules in cutaneous mucosal defence, we compared their transcriptional changes in salmon skin and spleen infected with the ectoparasite Lepeophtheirus salmonis which targets skin and mucus of salmonid fish (sampled 3, 14 and 28 days post infection (dpi)). Salsal pIgR and Salsal pIgRL transcripts significantly increased after 14dpi in skin and spleen. CLM1 was up-regulated in skin and down-regulated in spleen, possibly indicating that CLM1 expressing cells had migrated to the target site. Homology modeling using human pIgR domain 1 (PDB 1xed) identified structurally equivalent residues on both Salsal pIgR and Salsal pIgRL, and the same domain disulphide bridge topology. Cysteines 42 and 50 (IMGT numbering) are 7 residues apart in all V domains of Salsal pIgR, Salsal pIgRL, and mammalian [D1]. [Copyright &y& Elsevier]

Abstract: Two members, Bright/ARID3A and Bdp/ARID3B, of the ARID (AT-Rich Interaction Domain) transcription family are distinguished by their ability to specifically bind to DNA and to self-associate via a second domain, REKLES. Bright and Bdp positively regulate immunoglobulin heavy chain gene (IgH) transcription by binding to AT-rich motifs within Matrix Associating Regions (MARs) residing within a subset of VH promoters and the Eμ intronic enhancer. In addition, REKLES provides Bright nuclear export function, and a small pool of Bright is directed to plasma membrane sub-domains/lipid rafts where it associates with and modulates signaling of the B cell antigen receptor (BCR). Here, we characterize a third, highly conserved, physically condensed ARID3 locus, Brightlike/ARID3C. Brightlike encodes two alternatively spliced, SUMO-I-modified isoforms that include or exclude (Δ6) the REKLES-encoding exon 6. Brightlike transcripts and proteins are expressed preferentially within B lineage lymphocytes and coordinate with highest Bright expression in activated follicular B cells. Brightlike, but not BrightlikeΔ6, undergoes nuclear-cytoplasmic shuttling with a fraction localizing within lipid rafts following BCR stimulation. Brightlike, but not BrightlikeΔ6, associates with Bright in solution, at common DNA binding sites in vitro, and is enriched at Bright binding sites in chromatin. Although possessing little transactivation capacity of its own, Brightlike significantly co-activates Bright-dependent IgH transcription with maximal activity mediated by the unsumoylated form. In sum, this report introduces Brightlike as an additional functional member of the family of ARID proteins, which should be considered in regulatory circuits, previously ascribed to be mediated by Bright. [Copyright &y& Elsevier]

Abstract: In addition to modulate B cells function, B cell activating factor belonged to TNF family (BAFF) also regulates T cells response via BAFFR and transmembrane activator and calcium modulator and cyclophilin–ligand interactor (TACI) expressing on activated T cells. This study explored the effect of a recombinant fusion protein containing the extracellular ligand-binding portion of TACI and the Fc portion of human immunoglobulin G (TACI-Ig) on activated T cells that were obtained from antigen-specific T-cell responses mice model induced by keyhole limpet haemocyanin (KLH), the characteristics of KLH-challenged mice were observed simultaneously. KLH immunization leaded to a significant positive relationship between BAFF level in serum and the extent of spleen histopathology. Serum concentration of BAFF, APRIL, IgM and IgG antibodies to KLH, and IL-4 were increased under KLH immunization, but IL-2 synthesis was decreased, resulting in a downregulation of IL-2/IL-4 ratio. Antigen-specific T cells proliferation, IL-5 production, the percentage of Th and activation T cells were significantly upregulated, however, IL-2 secretion and the percentage of naïve T cells were downregulated in vitro. RhBAFF co-stimulation further evoked T cells hyperplasy, IL-4 and IFN-γ expression, the subgroups of Th, early antigen activation and activation T cells were also further increased. On the contrary, naïve T cells were further reduced under rhBAFF stimuli. Administration of rhTACI-Ig significantly inhibited T cells proliferation, cytokines production and T cells differentiation, and the inhibitory effects might be associated with its ability to neutralize both the exogenous and endogenetic BAFF and APRIL. [Copyright &y& Elsevier]

Abstract: Targeted delivery of antigen improves immunogenicity and can obviate the use of adjuvants. In addition to molecular targeting based on affinity interactions, particle-based antigen targeting to myeloid cells is also an efficient means to enhance immune responses. We compared the efficiency of targeting a model antigen, streptavidin, to CD40 and low affinity Fc gamma receptors II and III, either in a soluble or in a particulate form. Single chain fragments targeting these receptors were used to generate soluble tetramers with streptavidin or to decorate streptavidin coated nanobeads, and mice were immunized with the different formulations. Whereas particulate presentation of streptavidin enhanced total IgG1 and IgG2a levels, overall antigen specific antibody production increased in the case of targeted soluble antigen only, as assessed by reverse protein arrays and ELISPOT. In particular, soluble CD40 targeted antigen induced the strongest IgG2a responses, suggesting a Th1 bias compared to FcgammaRII/III targeting. Combined targeting to these receptors did not further increase immunogenicity. Thus, in our model, affinity targeting of soluble antigen to CD40 proved to be superior to particle-mediated delivery both in terms of antibody quantity and quality. [Copyright &y& Elsevier]

Abstract: B cells play important roles in normal immunity and human disease. While much has been learned from elegant studies utilizing genetically modified mice or with immunotherapy in normal and in autoimmune mice, we are merely at the start of learning about the mechanism(s) by which B cells contribute to human autoimmune disease. Here, I will review the most recent insights obtained with immunomodulatory therapies in human disease highlighting the clinical and immunological lessons we have learned with B cell depleting (e.g., rituximab) and immunomodulatory (e.g., BAFF/BLys neutralization through belimumab) therapies. [Copyright &y& Elsevier]

Abstract: Upon antigen stimulation, B lymphocytes differentiate into antibody secreting cells (ASC), most of which undergo apoptosis after a few days of intense Ig production. Differentiation entails expansion of the endoplasmic reticulum (ER) and requires XBP1 but not other elements of the unfolded protein response, like PERK. Moreover, normal and malignant ASC are exquisitely sensitive to proteasome inhibitors, but the underlying mechanisms are poorly understood. Here we analyze the role of C/EBP homologous protein (CHOP), a transcription factor mediating apoptosis in many cell types that experience high levels of ER stress. CHOP is transiently induced early upon B cell stimulation: covalent IgM aggregates form more readily and IgM secretion is slower in chop −/− cells. Despite these subtle changes, ASC differentiation and lifespan are normal in chop −/− mice. Unlike fibroblasts and other cell types, chop −/− ASC are equally or slightly more sensitive to proteasome inhibitors and ER stressors, implying tissue-specific roles for CHOP in differentiation and stress. [Copyright &y& Elsevier]

Abstract: The non-classical HLA-Ib molecule, HLA-E share several peptide sequence similarities with the heavy chains of classical HLA class Ia (-B and -C) molecules. Therefore, the antibodies to HLA-E, that recognize shared sequences, may bind to HLA-Ia alleles. This hypothesis is tested by examining the affinity of HLA-E monoclonal antibodies (HLA-E-MAbs) to HLA-Ia molecules and by inhibiting the antibody binding to both HLA-E and HLA-Ia with the shared peptide sequence(s). Single recombinant HLA molecule-coated beads are used for antibody binding. The antibody binding is evaluated by measuring mean fluorescence index [MFI] with Luminex multiplex flow-cytometric technology. The peptide-inhibition experiments are carried out with synthetic shared peptides, most prevalent to HLA-E and HLA-Ia alleles. The number of HLA-Ia alleles recognized by the HLA-E-MAbs varies with the density of the antigen (quantity of antigen-coated beads) and dilution of MAb. Binding of HLA-E-MAbs to β2 microglobulin (β2m)-free HLA-Ia antigens confirms the location of the epitopes on the heavy chain (HC) of the antigens. Strikingly, the nature of alleles of HLA-Ia recognized by different HLA-E-MAbs is identical. The binding of HLA-E-MAbs to the HLA-Ia is inhibited dosimetrically by the adjacent peptides, 115QFAYDGKDY123 and 137DTAAQI142, but not by 126LNEDLRSWTA135, another closer shared peptide sequence. The inhibitory peptide sequences in HLA-E are at the α2-helix terminal facing β2m. The HLA-Ia alleles recognized by HLA-E-MAb (e.g., MEM-E/02) are similar to those recognized by the natural anti-HLA antibodies found in the sera of healthy non-alloimmunized males. This study postulates that some, if not all, of the natural HLA-Ia antibodies seen in healthy males could be anti-HLA-E antibodies cross-reacting with HLA-Ia alleles. [Copyright &y& Elsevier]

Abstract: Domain antibodies (dAbs) are promising candidate therapeutics and diagnostics. Efficient selection of novel potent dAbs with potential for clinical utility is critically dependent on the library diversity and size, and the scaffold stability. We have previously constructed a large (size ∼2.5×1010) dAb library by grafting human antibody heavy chain complementarity determining regions (CDRs) 2 and 3 (H2s, H3s) into their cognate positions in a human heavy chain variable domain (VH) scaffold and mutagenizing the CDR1 (H1). High-affinity binders against some antigens were selected from this library but panning against others was not very successful likely due to limited diversity. We have hypothesized that by grafting highly variable, both in length and composition, human CDRs into non-cognate positions, the dAb library diversity could be significantly increased and the library would allow for more efficient selection of high-affinity antibodies against some targets. To test this hypothesis we designed a novel type of dAb library containing CDRs in non-cognate positions. It is based on our previous library where H1 was replaced by a library of human light chain CDR3s (L3s) thus combining three most diversified fragments (L3, H3 and H2) in one VH scaffold. This large (size ∼1010) phage-displayed library was highly diversified as determined by analyzing the sequences of 126 randomly selected clones. Novel high-affinity dAbs against components of the human insulin-like growth factor (IGF) system were selected from the new library that could not be selected from the previously constructed one. Most of the newly identified dAbs were highly soluble, expressible, monomeric and may have potential as candidate cancer therapeutics. The new library could be used not only for the selection of such dAbs thus complementing existing libraries but also as a research tool for the exploration of the mechanisms determining folding and stability of human antibody domains. [Copyright &y& Elsevier]

Abstract: N-Glycolylated (NeuGc) gangliosides are tumor-specific antigens and as such represent attractive targets for cancer immunotherapy. The chimeric antibody chP3 selectively recognizes a broad variety of NeuGc gangliosides, showing no cross-reactivity to the highly similar N-acetylated (NeuAc) gangliosides that are common cellular antigens in humans. Here, we report the crystal structure of the chP3 Fab and its computer-docking model with the trisaccharide NeuGcα3Galβ4Glcβ, which represents the carbohydrate moiety of the tumor-antigen NeuGc-GM3. The interaction involves only the heavy chain of the chP3 antibody. The modelled complex is consistent with all available experimental data and shows good surface complementarity. The negatively charged sialic acid residue NeuGc is buried in a pocket flanked by two arginine residues, VH Arg31 and VH Arg100A. We have further investigated the interaction of chP3 with its anti-idiotypic antibody, 1E10 (also known as Racotumomab), currently in clinical trials as a cancer vaccine. While many of the chP3 residues predicted to interact with the NeuGc ganglioside also feature prominently in the modelled complex of chP3 and 1E10, we do not observe structural mimicry. Rather, we suspect that the anti-idiotype 1E10 may serve as an imprint of the structural characteristics of the chP3 idiotype and, consequently, give rise to antibodies with P3-like properties upon immunization. [Copyright &y& Elsevier]

Abstract: Bioengineering of Galβ3GalNAcα, known as Thomsen–Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galβ3GalNAcα-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlαTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope. Antibodies produced by glycan bioengineering recognize HT29, T47D, MCF7, and CT26 epithelial tumor cells. Epithelial tumor cell adhesion to T antigen-binding lectins and endothelial cells was lower in the presence of antibodies raised against the engineered immunogen. The immune response directed to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens for immunotargeting purposes. [Copyright &y& Elsevier]

Abstract: The number of anti-cancer antibodies in therapy and in clinical trials is increasing gradually while their curative efficacy remains rather limited due to the resistance of tumor cells to complement-dependent cytotoxicity (CDC). An updated review of the various defense mechanisms complement is confronting when tackling a tumor cell is presented. The mechanisms discussed are: membrane and secreted complement regulatory proteins, heat shock proteins, extracellular proteases and protein kinases, cell surface sialylation and intracellular survival anti-lytic signals. Projected treatment strategies are depicted for each of the complement resistance mechanisms. It is conceivable that the therapeutic capacity of anti-cancer antibodies will be amplified once combined with a reagent that sensitizes the cancer cells to CDC. [Copyright &y& Elsevier]

Abstract: Intravenous drug use has become the principal route of hepatitis C virus (HCV) transmission due to the sharing of infected needles. In this study, we analyzed the distribution of HLA–KIR genotypes among 160 Puerto Rican intravenous drug users (IDUs) with HCV infection and 92 HCV-negative Puerto Rican IDUs. We found a significant association between the presence of different combinations of KIR inhibitory receptor genes (KIR2DL2 and/or KIR2DL3, pC=0.01, OR=0.07; KIR2DL2 and/or KIR2DL3 + KIR2DS4, pC=0.01, OR=0.39) and HLA-C1 homozygous genotypes (HLA-C1 + KIR2DS4, pC=0.02, OR=0.43; HLA-C1 + KIR2DL2 + KIR2DS4, pC=0.02, OR=0.40) together with the activating receptor KIR2DS4 (HLA-C1+ KIR2DS4 + KIR2DL3 and/or KIR2DL2, pC=0.004, OR=0.38) with protection from HCV infection. Our findings in HCV-infected and non-infected IDUs suggest an important role for KIRs (KIR2DL2 and KIR2DL3) with group HLA-C1 molecules, in the presence of activating KIR2DS4, in protection from HCV infection. These results support the hypothesis that activator signaling, mediated by KIR2DS4, plays a determinant role in the regulation of NK cell antiviral-activity. [Copyright &y& Elsevier]

Abstract: Therapeutic approaches aimed at targeting tumor surface markers using monoclonal antibodies provide a powerful strategy in cancer treatment. Here we report selection of single variable domains (VHH) of llama heavy chain antibodies, using a VHH-phage-display library. A reverse proteomic approach was used to identify the cognate proteins recognized by enriched VHH on HeLa cells. One of these VHH bound the integrin α3β1 (VLA-3) and was further characterized. Most interestingly, this VHH could inhibit VLA-3 mediated cell-matrix adhesion. Our approach provides a fast and efficient method to screen for novel cell surface markers on normal and tumor cells that may find diagnostic or therapeutic application in disease management or treatment. [Copyright &y& Elsevier]

Abstract: In the present report, we revisited the B cell responsiveness of 7 wild-derived mouse strains to various toll-like receptor ligands (TLR-L). We found that 2 of them, namely PWK and STF presented profound defects in B cell proliferative responses to most of the TLR-L. Yet, their macrophage responses were largely unaffected, suggesting that regulation of TLR pathways are distinct in B cells and macrophages. We also showed that, anti-CD40 mAbs rescued the low proliferative responses to CpG in both PWK and STF B cells. In the other hand, CpG synergized with LPS to induce high levels of proliferation in STF B cells, which did not respond to LPS alone. Cytokine or immunoglobulin (Ig) productions, in vitro, were less impaired than the proliferative responses to LPS or CpG alone. In STF B cells, both ERK, P38 and JNK pathways were affected following in vitro TLR4 or TLR9 signaling. Moreover, while the basal levels of Ig secreting cells and of serum Igs were similar to that of control mice, antibody responses to both TI and TD antigens were severely affected, mainly in STF mice. Our findings therefore highlight the relevance of wild-derived mouse strains and TLR-L to study B cell physiology. [Copyright &y& Elsevier]

Abstract: The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk−/− mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk−/− mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk−/− mice. A transgene expressing a low level of Btk in B cells (Btklo) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btklo B cells. anti-DNA IgG was not observed in 56R.Btklo mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached. [Copyright &y& Elsevier]

Abstract: The E3/19K protein of human adenovirus type 2 (Ad2) was the first viral protein shown to interfere with antigen presentation. This 25kDa transmembrane glycoprotein binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), thereby preventing transport of newly synthesized peptide–MHC complexes to the cell surface and consequently T cell recognition. Recent data suggest that E3/19K also sequesters MHC class I like ligands intracellularly to suppress natural killer (NK) cell recognition. While the mechanism of ER retention is well understood, the structure of E3/19K remains elusive. To further dissect the structural and antigenic topography of E3/19K we carried out site-directed mutagenesis and raised monoclonal antibodies (mAbs) against a recombinant version of Ad2 E3/19K comprising the lumenal domain followed by a C-terminal histidine tag. Using peptide scanning, the epitopes of three mAbs were mapped to different regions of the lumenal domain, comprising amino acids 3–13, 15–21 and 41–45, respectively. Interestingly, mAb 3F4 reacted only weakly with wild-type E3/19K, but showed drastically increased binding to mutant E3/19K molecules, e.g. those with disrupted disulfide bonds, suggesting that 3F4 can sense unfolding of the protein. MAb 10A2 binds to an epitope apparently buried within E3/19K while that of 3A9 is exposed. Secondary structure prediction suggests that the lumenal domain contains six β-strands and an α-helix adjacent to the transmembrane domain. Interestingly, all mAbs bind to non-structured loops. Using a large panel of E3/19K mutants the structural alterations of the mutations were determined. With this knowledge the panel of mAbs will be valuable tools to further dissect structure/function relationships of E3/19K regarding down regulation of MHC class I and MHC class I like molecules and its effect on both T cell and NK cell recognition. [Copyright &y& Elsevier]

Abstract: The systemic inflammatory response syndrome (SIRS) is triggered by C5a generation following an excessive complement amplification, but it has remained unclear how complement amplification is stimulated. It is known that neutrophilic elastase can cleave IgG to F(ab′)2 and that F(ab′)2-containing immune complexes (F(ab′)2-IC) stimulate complement amplification together with an unidentified plasma factor. We show that absorption of plasma on F(ab′)2 from human IgG removed this factor and prevented F(ab′)2-IC from stimulating complement amplification. The required factor was purified from pooled whole human IgG (IVIG) as those naturally occurring antibodies (NAbs) that bind to F(ab′)2, but not to intact IgG. These “anti-hinge NAbs” restored complement amplification by F(ab′)2-IC in absorbed plasma. Anti-hinge NAbs must have formed secondary, rigidified IC from F(ab′)2-IC, because the F(ab′)2 fragments evidently captured dimeric C3b, known as a potent C3 convertase precursor. This process may also stimulate complement amplification in vivo, because plasma from septic patients at the onset of SIRS indeed contained F(ab′)2 fragments. The concentrations of F(ab′)2 and that of factor Bb, an unbiased measure of complement amplification, correlated linearly with that of released elastase. Moreover, the F(ab′)2 fragments migrated on gelfiltration columns together with anti-hinge NAbs as ICs with MW of up to ∼750kDa, as verified on plasma of each of the nine patients studied. These findings provide for the first time a plausible mechanism of how F(ab′)2-containing immune complexes stimulate complement amplification together with anti-hinge NAbs. The same mechanism may contribute to complement overreaction at the onset of SIRS. [Copyright &y& Elsevier]

Abstract: The murine single-chain variable fragment F8 (scFv(F8)) is endowed with high intrinsic thermodynamic stability and can be functionally expressed in the reducing environment of both prokaryotic and eukaryotic cytoplasm. The stability and intracellular functionality of this molecule can be ascribed mostly to its framework regions and are essentially independent of the specific sequence and structure of the supported antigen-binding site. Therefore, the scFv(F8) represents a suitable scaffold to construct stable scFv chimeric molecules against different antigens by in vitro evolution or antigen-binding site grafting. Thanks to the favourable pharmacokinetic properties associated to a high thermodynamic stability of antibody fragments, such scFv(F8) variants may be exploited for a wide range of biomedical applications, from in vivo diagnosis to therapy, as well as to interfere with the function of intracellular proteins and pathogens, and for functional genomics studies. However, the potential immunogenicity of the murine framework regions represents a limitation for their exploitation in therapeutic applications. To overcome this limitation, we humanized a derivative of the scFv(F8), the anti-lysozyme scFv(11E), which is endowed with even higher thermodynamic stability than the parent antibody. The humanization was carried out by substituting the framework residues differing from closely related VH and VL domains of human origin with their human counterparts. Site-directed mutagenesis generated the fully humanized product and four intermediate scFvs, which were analyzed for protein expression and antigen binding. We found that the substitution Tyr 90→Phe in the VH domain dramatically reduced the bacterial expression of all mutants. The back-mutation of Phe H90 to Tyr led to the final humanized variant named scFv(H5)H90Tyr. This molecule comprises humanized VH and VL framework regions and is endowed with HEL-binding affinity, stability in human serum and functionality under reducing conditions comparable to the murine cognate antibody. Consequently, the humanized scFv(H5)H90Tyr represents a suitable scaffold onto which new specificities towards antigens of therapeutic interest can be engineered for biomedical applications. [Copyright &y& Elsevier]

Abstract: The active component of the licensed human anthrax vaccine (BioThrax™, or AVA) is a Bacillus anthracis toxin known as protective antigen (PA). Second generation anthrax vaccines currently under development are also based on a recombinant form of PA. Since the current and future anthrax vaccines are based on this toxin, it is important that the immunobiology of this protein in vaccinated humans be understood in detail. We have isolated and analyzed the PA-specific antibody repertoire from an AVA-vaccinated individual. When examined at the clonal level, we find an antibody response that is complex in terms of the combinatorial elements and immunoglobulin variable genes employed. All PA-specific antibodies had undergone somatic hypermutation and class switch recombination, both signs of affinity maturation. Although the antigenic epitopes recognized by the response were distributed throughout the PA monomer, the majority of antibodies arising in this individual following vaccination recognize determinants located on the amino-terminal (PA20) sub-domain of the molecule. This latter finding may have implications for the rational design of future PA-based anthrax vaccines. [Copyright &y& Elsevier]

Abstract: The antigen-binding site, the paratope, of an antibody can be seen as being composed of a central core and a more peripheral area situated at its rim. Naturally these regions acquire their diversity using different mechanisms and they also have dissimilar roles, as they contribute differently to the binding interaction. Also, antigens of different size utilize these regions differently; while haptens mainly interact with the central core, larger antigens have additional interactions in more peripheral regions. Since haptens do not occupy the entire available paratope we hypothesized that hapten-specific antibodies, as they develop naturally or in the laboratory, have an imprint of the carrier protein they were once selected on. By using combinatorial library and phage display technologies on a hapten-specific antibody we were able to demonstrate that a peripheral carrier imprint indeed exists. We further show that such an imprint can act as a seed in the evolution of binders that recognize the carrier protein even in the absence of the hapten modification. The observed results provide a plausible mechanism for how haptenization of self-antigens can lead to the development of autoimmunity. [Copyright &y& Elsevier]

Abstract: The anti-C1q antibodies present in systemic lupus erythematosus (SLE) patients’ sera are associated with renal involvement and the titer of these autoantibodies correlates with the clinical activity of the disease. It has previously been shown that anti-C1q antibodies bind neo-epitopes within the collagen region of human C1q. Evidence that these polyclonal autoantibodies recognize epitopes within the globular domain (gC1q) of the molecule has not been documented. In this study, we screened, using ELISA, a number of sera from SLE patients for the presence of anti-gC1q autoantibodies using recombinant globular head regions of individual A (ghA), B (ghB) and C (ghC) chains of human C1q. The recombinant proteins were used as test antigens to determine the levels of autoantibodies directed against ghA, ghB and ghC. SLE sera, containing high levels of anti-C1q antibodies, showed differentially increased binding towards ghA and ghB, which suggested that the gC1q domain can also be target of anti-C1q antibodies generated in SLE patients. Such antibodies can have severe pathophysiological consequences since these are likely to further impair the ability of C1q to clear immune complexes. [Copyright &y& Elsevier]

Abstract: Inhibitory antibodies directed against blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic and autoimmune patients. Identifying B-cell FVIII epitopes and mapping them on the molecule remain important challenges. Using a combination of different algorithms, more than 30 hypothetical linear epitopes were predicted on the FVIII molecule surface. We selected several major predicted sequences, spanning all FVIII domains, for specific antibody induction in rabbits. All peptides tested successfully induced production of specific anti-FVIII rabbit antibodies, supporting the relevance of our approach. To investigate the presence of FVIII-reactive antibodies in the healthy donor population, a pooled fraction rich in all IgG subclasses was purified on peptide-Sepharose columns. Substantial amounts of Ig, specific for each FVIII peptide, were purified with yields ranging from 8 to 223ng/mg immunoglobulins. Our results confirm the diversity of FVIII epitopes recognised by natural human anti-FVIII autoantibodies. All IgG subclasses were found in the affinity-isolated anti-peptide material, with overrepresentation of IgG2 and IgG4. Evidence was also found for new FVIII epitopes. Five human anti-peptide preparations displayed FVIII-neutralising activity, ranging from 1.3 to 5.3BU/mg. Although the presence of naturally occurring anti-FVIII antibodies in healthy donors has been previously described, our methodology has allowed, for the first time, a fine mapping of several inhibitory and non-inhibitory epitopes. Our observations support the hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones. [Copyright &y& Elsevier]

Abstract: The presence of autoantibodies to C1-inhibitor (C1-INH-Abs) is a hallmark of acquired C1-inhibitor deficiency. However, only scarce data are available on their prevalence in hereditary angioedema (HAE). In a prospective study performed between 2001 and 2004 in 95 patients with Type I or II HAE, serum samples were taken one to three times a year and clinical status of the patients was registered. Serum samples were tested for total activity of the classical pathway, C1q, C3, C4 and C1-inhibitor (C1-INH) concentration and activity levels, as well as the presence of IgG, IgA and IgM type anti-C1-inhibitor antibodies (C1-INH-Ab). Fifty-four healthy age and gender matched persons served as control. Significant differences between the patients and controls in the occurrence of elevated (2S.D. higher than mean of control) C1-INH-Abs titers was found only in the case of IgM type C1-INH-Abs. Elevated (>4.22AU/ml) IgM C1-INH-Abs levels were found in 31 and 4% of the patients and controls, respectively (p <0.001). Surprisingly, high titer IgM C1-INH-Abs were present with equal frequency in the 41 HAE patients ever treated with C1-INH concentrate and in the 54 C1-INH treatment naïve patients. In the latter group, strong positive correlation between the levels of the IgM C1-INH-Abs and the most severe disease (score 1) (p =0.0021) and the yearly attack rate (p =0.0173) were obtained. In addition, the levels of the IgM C1-INH-Abs exhibited strong negative correlation to the C1-inhibitor concentration and functional activity, total classical complement pathway activity, and a positive correlation to total IgM concentration. Taken together, these data indicate that IgM type C1-INH-Abs are present with highly elevated frequency in HAE patients irrespectively of the previous treatment with C1-INH concentrate. Most probable production of these autoantibodies is the consequence of the activation of complement and other plasma enzyme systems during HAE attacks. Determination of IgM C1-INH-Abs can be used as an activity marker in HAE. [Copyright &y& Elsevier]

Abstract: Oxidized low-density lipoprotein (oxLDL) is a key autoantigen in atherosclerosis. The genetic structures and pathogenic roles of autoantibodies against this protein remain to be established. In this study, we cloned several monoclonal IgG autoantibody Fab fragments specific for oxLDL from peripheral blood lymphocytes of atherosclerosis patients, using phage display technology. The sequences of their variable regions were determined at the cDNA level. The closest germline counterparts for the heavy chains belonged to the VH3 or VH1 family. The sequences and lengths of complementarity-determining regions (CDR)3-VH were diverse, and frequent mutations of positively charged amino acids (particularly arginine) over entire VH and VL sequences were observed. It is proposed that anti-oxLDL autoantibody formation is driven by antigens. Among the Fabs, P2-8 and P3-175 bound to both MDA-LDL and Cu-oxLDL, and inhibited the uptake of oxLDL by macrophages, suggesting the epitope(s) recognized by the Fabs is a part of ligands on oxLDL that is involved in uptake by macrophage scavenger receptor. These human autoantibody Fabs require detailed investigation to ascertain their potential as agents for clinical applications. [Copyright &y& Elsevier]

Abstract: RNA interference (RNAi), a process that specifically silences target gene expression, is a powerful technique to modulate cellular functions. In this study, we identified two small interference RNA (siRNA) sequences that can specifically and efficiently silence the expression of the granulocyte colony-stimulating factor receptor (GCSF-R) gene and achieved stable knockdown of GCSF-R using pFIV lentivirus containing the GCSF-R siRNA. GCSF-R knockdown significantly reduces the expression of IL-lα, IL-lβ, IL-6, IL-10, H-2Kb, I-Ab, CD80 and CD86, and increases PDL1 and PDL2 expression, while IL-12p35, TGFβ, TNFα and CD40 expression is unaltered. Furthermore, GCSF-R knockdown significantly changes the endocytosis and micro-pinocytosis abilities as well as surface expression of antigens of DC2.4 cells. [Copyright &y& Elsevier]

Abstract: Selection of specific antibodies from large repertoires is of importance in generating antibodies to specific structural determinants and in studying structure–function relationships. Alkaline phosphatase (AP) has several isozymes with various degrees of homology and a range of common synthetic substrates. We have previously reported the generation of isozyme specific anti-enzyme antibodies to an oncofetal antigen, placental alkaline phosphatase (PLAP) by using a specific uncompetitive inhibitor, l-Phe-Gly-Gly along with the substrate para-nitrophenyl phosphate (pNPP), to elute scFvs from a phage-displayed immunoglobulin library. These antibodies were directed to the active site and inhibited enzyme activity. An uncompetitive inhibitor acts by stabilizing the enzyme–substrate (ES) complex. In the present work, we report the characteristics of a clone VE5, selected by the same method. This clone has a higher binding affinity for ES complex than for enzyme alone. This is true for all the three isozymes (placental, bone and intestinal) tested. However, the other synthetic small molecular substrate, disodium phenyl phosphate inhibits phage binding. The clone possibly binds to the conserved structures of the active site of the AP isozymes and the higher affinity binding to AP–pNPP complex reflects the method of selection. Such anti-enzyme antibodies have a possible potential role in dissecting structure–function relationship of enzymatic antigens. [Copyright &y& Elsevier]

Abstract: A study aiming at cloning and characterizing natural antibodies to human immunodeficiency virus type 1 (HIV-1) targets is described. In particular, we report the molecular cloning of a Fab molecule binding the HIV-1/Tat protein from a seronegative patient. The Fab was characterized for its binding specificity and investigated in regards to its molecular structure. Furthermore, to evaluate the role played by the heavy and light chains in the binding to the antigen, hybrid Fabs were constructed combining the heavy and the light chain of the natural anti-Tat clone with a control high-affinity Fab derived from the repertoire of the same patient. The results indicate that the natural immunoglobulin under study: (i) is a polyreactive antibody of IgG1 isotype, and not an IgM as usually described for anti-HIV natural clones, (ii) shows a pattern of mutations compatible with an antigen-driven mechanisms, (iii) its heavy chain derives from a V-gene subfamily (V3-23) highly represented in fetal life, and (iv) its heavy chain variable region exhibits several characteristics, including an extremely long, hydrophilic CDR3, that are unusual and theoretically important in determining the polyreactive capacity of the molecule. [Copyright &y& Elsevier]

Abstract: Analysis of the N-linked oligosaccharides of the murine IgG1 monoclonal antibody (mAb) to Cryptococcus neoformans by LC/MS revealed five different core fucosylated, biantennary complex-type oligosaccharides at Asn-293, with the major species being a mono-galactosylated oligosaccharide with the glycosyl composition of Hex4HexNAc4Fuc (39% of the total glycan pool). The primary sequence predicted from nucleic acid sequencing differed from that measured by mass spectrometry at position 33 (ASN to ASP), a finding that may represent post-translational modification caused by spontaneous ASP deamination. Analysis of mAb 18B7 from three hybridoma clones revealed the same heterogenous N-glycan pattern, indicating that diversity in oligosaccharide structures originated from individual cells. The binding of native and de-glycosylated mAb 18B7 to cryptococcal Ag was comparable but the de-glycosylated 18B7 had shorter serum half-life and did not activate complement (C). De-glycosylated mAb 18B7 was opsonic for C. neoformans with murine macrophages through a mechanism that involved C-independent ingestion through the C receptor. Passive administration of de-glycosylated mAb 18B7 mediated comparable protective efficacy to the native mAb in mice with lethal infection. The results imply that the contribution of N-glycan structure to immunoglobulin function varies depending on the Ag–Ab system. [Copyright &y& Elsevier]

Abstract: In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens. [Copyright &y& Elsevier]

Abstract: Although a rich database of Igh a allotype mouse immunoglobulin germline genes exists, current information on Igh b allotype immunoglobulin germline genes is limited. Among the immunoglobulin VH genes, single-cell amplified from six Igh b (C57BL/6 background) spleens in this study, 602 clonally independent immunoglobulin VH sequences belonging to the VH1/J558 family were identified. Whereas 335 of these sequences could be traced to have originated from 29 different VH1/J558 germline genes deposited in the NCBI Igblast database, the remaining 267 sequences appeared to have originated from 21 novel germline genes. Of the 50 VH1/J558 germline genes utilized in the peripheral repertoire of these Igh b allotype mice, the most frequently used genes included 45.21.2, V165.1, J558.6, J558.18A, and V23. Whereas the majority of the novel genes uncovered represented allelic counterparts of previously described Balb/c (Igh a allotype) genes, some appeared to represent truly novel germline genes. Collectively, the VH1/J558 germline genes exhibited high amino acid residue usage variability at the CDR1 positions, H31, H33, and H35, and the CDR2 positions, H50, H52, H53, H54, H56, and H58. The 50 VH1/J558 germline genes expressed in the peripheral Igh b repertoire also varied widely in the net charge of their CDR regions, raising the possibility that they may be differentially utilized to encode anti-nuclear autoantibodies. [Copyright &y& Elsevier]

We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein β2-Glycoprotein I (β2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to β2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to β2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-β2-GPI activity. Furthermore, there was a low increase (≅10%) in the binding of UK4 to cardiolipin in the presence of β2-GPI. The results demonstrate that anti-β2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of β2-GPI. Molecular docking experiments highlight a number of potential sites on β2-GPI for interaction of UK4 and indicate as to how β2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-β2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed. [Copyright &y& Elsevier]

The potential role of the regions (carboxil and amino) of the Cry proteins in the ability of these proteins to elicit strong immune responses was investigated. Intraperitoneal immunization of mice with the homologous Cry1A protoxins (130–133 kDa), with the long C-terminal half gave rise mostly to similar, strong serum and mucosal IgG and IgM antibody response but a lower induction of these Ab by intranasal route. Remarkably, Cry3A protoxin, devoid of C-terminal half was able to induce a significant mucosal IgG, and IgM Ab as well as Cry1A protoxins, suggesting us that immunogenic abilities are not restricted to C-terminal half but N-terminal half itself could be involved. In fact, this assumption was strengthen by the strong immunogenic abilities of the Cry1A toxins, specially IgG and IgA Ab induced by both routes in different mucosal sites. These data indicate that immunogenic abilities of the Bt Cry proteins reside and depends of the N-terminal half. [Copyright &y& Elsevier]

The fine binding characteristics of three well-characterized human autoantibodies B3, RH14 (anti-DNA) and UK4 (anti-cardiolipin) in their IgG and cloned Fab formats, were investigated. Although in severe combined immunodeficiency (SCID) mice B3 and RH14 both induce proteinuria, only RH14 induces early features of lupus nephritis, whereas UK4 exhibits lupus anticoagulant activity. RH14 exhibited up to 10 fold higher binding to DNA compared to that shown by B3 or UK4 and involved significant electrostatic and phosphate group interactions. Only RH14 exhibited strong anti-Sm cross-reactivity residing on the C-terminus of the antigen as determined by the use of 76 overlapping 15mer peptides. Chain shuffling experiments indicate that anti-Sm/RNP and anti-Jo-1 activities of B3 and UK4 co-exist on one of the two chains (light, B3; heavy, UK4). The present study provides evidence that a human anti-DNA antibody can also be an anti-ENA antibody. Furthermore, the anti-DNA antibodies also exhibited cross-reactivity against glutathione-S-transferase and DNA polymerase PolIV of bacterial origin. This is the first demonstration of the presence of such cross-reactivities on lupus anti-DNA antibodies. We now demonstrate that subsets of sera from the patients with lupus, recognise these antigens. This observation may in some cases provide a mechanism for the common expression of a variety of autoantibodies observed in systemic lupus erythematosus (SLE). [Copyright &y& Elsevier]

The functional valency of a monoclonal antibody (mAb) has important influences on such things as antigen avidity, Fc-mediated immune effector functions, and clearance of immune complexes. cV1q, a neutralizing rat/mouse chimeric anti-mouse tumor necrosis factor (TNF) monoclonal antibody (mAb), and Rt108, a neutralizing mouse anti-rat TNF (anti-raTNF) mAb, appear to be functionally monovalent for TNF-binding despite containing two antigen binding sites. The functional monovalency of these two independent anti-rodent TNF mAbs is presumably a result of steric hindrance from one TNF molecule binding to one Fab arm that prevents binding of a second TNF molecule to the other Fab arm. To test whether this steric hindrance could be overcome by introducing extra space and flexibility between the Fab arms, these mAbs were engineered to contain an extra CH1 immunoglobulin domain between the CH1 and hinge domains of their heavy chains. In vitro binding data showed that, compared to the original mAbs, the modified mAbs (S-mAbs) had greater capability of binding two TNF molecules simultaneously. In vitro activity assays showed that, compared to the original mAbs, the S-mAbs had significantly greater TNF-neutralization potency, with the S-mAb version of cV1q (S-cV1q) being 200-fold more effective at blocking mouse TNF (muTNF) and the S-mAb version of Rt108 (S-Rt108) being 20-fold more effective at blocking raTNF. Similar results were observed in vivo, where S-cV1q was between 100- and 500-fold more protective than cV1q in mice challenged with endotoxin. These data reveal that introduction of another constant region immunoglobulin domain into two unrelated mAbs dramatically enhanced their neutralization potency. Other mAbs may also show more potent activity using this engineering approach, particularly mAbs that recognize homopolymeric antigens. [Copyright &y& Elsevier]

We prepared IgG and IgM with identical combining sites to a hapten, (4-hydroxy-3-nitrophenyl)acetic acid (NP), and used surface plasmon resonance to evaluate the association constants (Ka) in interactions of these antibodies (Abs) with antigens (Ags) which differed in the size of carriers and NP valence as well as in the stoichiometry of Ag to Ab in the immune complexes. It was found that IgM was unable to form an Ag1Ab1 complex with the highly haptenated Ag, NP18.6-bovine serum albumin (BSA), such that one NP18.6-BSA molecule was held by multiple contacts with Fab arms from five subunits, although IgM was capable of forming an Ag4Ab1 complex in which each subunit was bound to one NP18.6-BSA molecule. IgM was superior to IgG in interactions with large Ags of low hapten density. The Ka values of IgM to these Ags were estimated to be ∼1×109 M-1, about 20-fold higher than those of IgG. Reduction of inter-subunit and inter-chain disulfide bonds resulted in a decrease in Ka values to large Ags but no change in those to small Ags. [Copyright &y& Elsevier]