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

Liposomal Co-Entrapment of CD40mAb Induces Enhanced IgG Responses against Bacterial Polysaccharide and Protein

Background Antibody against CD40 is effective in enhancing immune responses to vaccines when chemically conjugated to the vaccine antigen. Unfortunately the requirement for chemical conjugation presents some difficulties in vaccine production and quality control which are compounded when multivalent vaccines are required. We explore here an alternative to chemical conjugation, involving the co-encapsulation of CD40 antibody and antigens in liposomal vehicles. Methodology/Principal Findings Anti-mouse CD40 mAb or isotype control mAb were co-entrapped individually in cationic liposomal vehicles with pneumococcal polysaccharides or diphtheria and tetanus toxoids. Retention of CD40 binding activity upon liposomal entrapment was assessed by ELISA and flow cytometry. After subcutaneous immunization of BALB/c female mice, anti-polysaccharide and DT/TT responses were measured by ELISA. Simple co-encapsulation of CD40 antibody allowed for the retention of CD40 binding on the liposome surface, and also produced vaccines with enhanced imunogenicity. Antibody responses against both co-entrapped protein in the form of tetanus toxoid, and Streptococcus pneumoniae capsular polysaccharide, were enhanced by co-encapsulation with CD40 antibody. Surprisingly, liposomal encapsulation also appeared to decrease the toxicity of high doses of CD40 antibody as assessed by the degree of splenomegaly induced. Conclusions/Significance Liposomal co-encapsulation with CD40 antibody may represent a practical means of producing more immunogenic multivalent vaccines and inducing IgG responses against polysaccharides without the need for conjugation

Galectin-9 Controls CD40 Signaling through a Tim-3 Independent Mechanism and Redirects the Cytokine Profile of Pathogenic T Cells in Autoimmunity

While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4loCD40+ effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4loCD40+ T cells. Understanding the dynamics of the CD40 receptor(s) and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones

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

Co-stimulatory agonists as immunological adjuvants

The considerable advances made in the fields of molecular biology, genomics, proteomics and protein engineering have led to the identification of a vast range of potential vaccine antigens for a host of man's most serious diseases. However, experience informs us that vaccines based on recombinant proteins and synthetic peptides lack the immunogenicity of the whole, killed pathogens used in traditional vaccines and, as such, clinical use of these immunogens remains negligible. In order to fully realize the potential benefits of recombinant antigen-based vaccines there is a pressing need to identify powerful adjuvants which can safely enhance these weak responses with a minimum of undesirable side effects. Adjuvant research represents a vibrant and fast moving field and recent developments suggest the goal of generating effective, safe and affordable ways of enhancing immune responses appears to be almost within our grasp. The purpose of this article is to review recent advances in adjuvant development using approaches that directly exploit the immune system's own co-stimulatory pathways to exert their function; with a particular emphasis on CD40 and CD28 based therapies

CD40 antibody as an adjuvant induces enhanced T cell responses

Monoclonal antibodies against CD40, conjugated to antigen, act as potent immunological adjuvants for primary antibody responses. We show here that CD40mAbs can also act as strong adjuvants for memory antibody responses, and for T cell responses as measured by ex vivo T cell proliferation to antigen, and delayed type hypersensitivity. Interferon gamma secretion in response to antigen is also enhanced. Finally, the adjuvant effect of CD40mAbs for secondary antibody responses is transferred with T cells rather than B cells. CD40mAb apparently have potent adjuvant effects on both Th1-like cells, and on T cells able to promote B cell antibody production. It is possible that the adjuvant effects of CD40 are mediated at least in part, indirectly, through enhanced antigen presentation by specific B cells, to T cells

Antibodies against cell surface antigens as very potent immunological adjuvants

We describe here two very potent adjuvant systems which are thought to work directly on antigen specific lymphocytes, thus by-passing the normal route for adjuvants, which is to activate antigen presenting cells (APCs) inducing release of inflammatory cytokines with resultant side effects of local and systemic reactogenicity. CD40 and CD28 based adjuvants are extremely potent and should avoid the inflammatory side effects induced by most adjuvants

CD40 antibody as a potent immunological adjuvant: CD40 antibody provides the CD40 signal to B cells, but does not substitute for T cell help in responses to TD antigens

Agonistic antibodies against CD40 have great potential as immunological adjuvants. We have shown that CD40mAbs induce strong antibody responses against conjugated antigen, and that this enhancement of responses extends to any sequence physically associated to the CD40 binding moiety, including the antibody's own Fc region. Thus, the CD40mAb acts as a model immunogen, containing both antigenic (i.e. Fc portion) and CD40 binding motifs (i.e. CD40 binding moiety). Using this system we examine here whether CD40mAb is able to directly mimic T cell help to B cells. CD40mAbs have no adjuvant effect in CD4 depleted mice, and thus, do not mimic T cell help. Simultaneous administration of recombinant IL-4 was unable to restore the adjuvant action of anti-CD40 in T cell depleted mice. However, CD40mAbs are effective adjuvants in CD154−/− mice, indicating that the antibodies are able to provide the CD40 stimulus to B cells which is naturally lacking in these mice. Identification of the additional stimuli required to fully mimic T cell help may be advantageous in vaccination of immunosuppressed patients