CD40mAb adjuvant induces a rapid antibody response that may be beneficial in post-exposure prophylaxis

Active vaccination can be effective as a post-exposure prophylaxis, but the rapidity of the immune response induced, relative to the incubation time of the pathogen, is critical. We show here that CD40mAb conjugated to antigen induces a more rapid specific antibody response than currently used immunological adjuvants, alum and monophosphoryl lipid A™

A Novel Redox Method for Rapid Production of Functional Bi-Specific Antibodies For Use in Early Pilot Studies

We demonstrate here a rapid alternative method for the production of functional bi-specific antibodies using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA). Following reduction of a mixture of two monoclonal antibodies with MESNA to break inter heavy chain bonds, this solution is dialysed under oxidising conditions and antibodies are allowed to reform. During this reaction a mixture of antibodies is formed, including parental antibodies and bi-specific antibody. Bi-specific antibodies are purified over two sequential affinity columns. Following purification, bi-specificity of antibodies is determined in enzyme-linked immunosorbent assays and by flow cytometry. Using this redox method we have been successful in producing hybrid and same-species bi-specific antibodies in a time frame of 6–10 working days, making this production method a time saving alternative to the time-consuming traditional heterohybridoma technology for the production of bi-specific antibodies for use in early pilot studies. The use of both rat and mouse IgG antibodies forming a rat/mouse bi-specific antibody as well as producing a pure mouse bi-specific antibody and a pure rat bi-specific antibody demonstrates the flexibility of this production method

NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival.

Neutrophil lifespan is plastic and highly responsive to factors that regulate cellular survival. Defects in neutrophil number and survival are common to both hematologic disorders and chronic inflammatory diseases. At sites of inflammation, neutrophils respond to multiple signals that activate protein kinase A (PKA) signaling, which positively regulates neutrophil survival. The aim of this study was to define transcriptional responses to PKA activation and to delineate the roles of these factors in neutrophil function and survival. In human neutrophil gene array studies, we show that PKA activation upregulates a significant number of apoptosis related genes, the most highly regulated of these being NR4A2 and NR4A3 Direct PKA activation by the site-selective PKA agonist pair N6/8-AHA and treatment with endogenous activators of PKA, including adenosine and PGE2, results in a profound delay of neutrophil apoptosis and concomitant upregulation of NR4A2/3 in a PKA dependent manner. NR4A3 expression is also increased at sites of neutrophilic inflammation in a human model of intradermal inflammation. PKA activation also promotes survival of murine neutrophil progenitor cells, and siRNA to NR4A2 decreases neutrophil production in this model. Antisense knockdown of NR4A2 and NR4A3 homologues in zebrafish larvae significantly reduces absolute neutrophil number without affecting cellular migration. In summary, we show that NR4A2 and NR4A3 are components of a downstream transcriptional response to PKA activation in the neutrophil, and that they positively regulate neutrophil survival and homeostasis

Redox method overview.

<p>MAb 1 and mAb 2 are mixed and reduced using MESNA, resulting in mvAb fragments being formed as visualized by SDS-PAGE. When dialysed into oxidising conditions mAbs are reformed resulting in a mixture of parental and bi-specific antibodies. In order to get a pure bsAb solution, antibodies are purified over two sequential affinity columns, each one specific for the one mAb only. After affinity column one the eluate containing mAb 1 and bsAb is purified on column 2. The resulting eluate contains pure bsAb that can be used for further applications.</p

Verification of antigen recognition by same species bsAbs.

<p>Same species bsAb was detected using a sandwich ELISA specific for biotin or DNP. Pre-labeling of G28/5 and 1C10 with DNP and A20 and GL117 with biotin allowed binding of 1C10-GL117 bsAb and G28/5-A20 bsAb and not parental antibodies in this assay (a and b respectively).</p

Redox method optimisation.

<p>Non-reducing SDS-PAGE showing the range of MESNA concentrations used in optimisation studies for GL117 mAb (a). Arrows indicate presence of whole mAb (1), mvAb (2), heavy chain (3) and light chain (4). 50 mM MESNA efficiently cleaves inter heavy chains in GL117 mAb resulting in a band at around 90 kDa (b) and in A20 mAb at around 95 kDa (c). Dialysis into PBS shows successful reformation of mvAb into whole mAb (d). Image shows in lane order; molecular weight standard, parental GL117 mAb and parental A20 mAb (both >170 kDa), MESNA-reduced GL117 and A20 mvAb mixture (90–110 kDa) and reformed whole antibody, including bi-specific GL117-A20 bsAb. Molecular weight (MW) marker PageRuler pre-stained protein ladder 10–170 kDa (Fermentas GmbH, St. Leon-Rot, Germany). Red.  =  Reducing conditions, Oxid.  =  Oxidising conditions.</p

Verification of antigen recognition by hybrid bsAbs.

<p>The presence of GL117-A20 bsAb (a) and 1C10-A20 bsAb (b) was verified by simultaneous recognition of anti-rat IgG and anti-mouse IgG in ELISA assay 1 as visualized by an increase in the OD reading at 450 nm compared to parental mAbs. Antigen recognition of β-gal and recognition by an anti-mouse IgG antibody was determined in ELISA assay 2 showing the presence of hybrid GL117-A20 bsAb after binding of the GL117 binding arm to β-gal and the A20 binding arm to anti-mouse IgG (c). CD40 specificity and subsequent recognition by an anti-mouse IgG antibody was determined in ELISA assay 3 and this showed the presence of 1C10-A20 bsAb by the binding of the 1C10-binding arm to CD40 and the A20 binding arm to anti-mouse IgG (d). Flow cytometric analysis on viable CD40L cells showed an increase in median fluorescence after incubation with 1C10-A20 bsAb but not GL117-A20 bsAb (e). Neg CTL  =  negative control.</p

Evidence of a functional B-cell immunodeficiency in adults who experience serogroup C meningococcal disease

After adolescence, the incidence of meningococcal disease decreases with age as a result of the cumulative immunizing effect of repeated nasopharyngeal colonization. Nevertheless, some adults succumb to meningococcal disease, so we hypothesized that this is due to a subtle functional immunological defect. Peripheral blood lymphocytes derived from survivors of serogroup C meningococcal disease and from age- and sex-matched controls were incubated with a polyclonal B-cell activator containing anti-immunoglobulin D (alpha-delta-dex) employed to mimic antigen-specific stimuli encountered during immune responses to bacterial polysaccharides, with and without T-cell activation (using anti-CD3/anti-CD28). Subsequent proliferation and activation of T and B lymphocytes were measured. In patients, T-cell responses to polyclonal stimuli and the delivery of T-cell help to B cells were unimpaired. Levels of B-cell proliferation in response to alpha-delta-dex stimulation alone were low in all samples but were significantly lower in patients than in controls, and these differences were more pronounced with the addition of T-cell help. The data are consistent with the presence of a subtle immunodeficiency in adults who have exhibited susceptibility to meningococcal disease. This defect is manifested as an impaired B-cell response to T-cell-independent type 2 antigens analogous to bacterial capsular polysaccharide

Contact dependent suppression of CD4 T cell activation and proliferation by B cells activated through IgD cross-linking

Although the co-stimulatory interaction between B and T cells is well defined, recent evidence suggests that B cells also have a regulatory role. Here, we show that B cells activated using anti-IgD conjugated to dextran (α-δ-dex) directly inhibit TCR-induced CD4 T cell activation, proliferation and cytokine production. This effect was observed in CD4 T cells activated both with and without CD28 co-stimulation. T cell viability was unaffected, and the T cell suppressive effect was mediated by contact with IgD activated purified B cells and not by IL-10 or other soluble factors. This is the first evidence of IgD activated B cells mediating inhibition of activation and proliferation of CD4 T cells in humans. This article is protected by copyright. All rights reserved.</p