Mucosal delivery of the human immunodeficiency virus-1 Tat protein in mice elicits systemic neutralizing antibodies,cytotoxic T lymphocytes and mucosal IgA

Human immunodeficiency virus (HIV)-1 Tat protein induces protection in non-human primates upon systemic vaccination. In view of the design of mucosal vaccines against HIV-1 we studied the immune response to native Tat (aa 1-86) in mice following intranasal delivery of the protein with two mucosal adjuvants, Escherichia coli heat-labile enterotoxin (LT) and LT-R72, a non-toxic mutant of LT. Immunization with Tat and the two adjuvants induced in BALB/c but not in C57BL/6 mice high and persistent levels of serum IgG and secretory IgA in vaginal and intestinal fluids. Mice sera neutralized Tat and recognized two epitopes mapping in the regions 1-20 and 46-60. Furthermore, their splenocytes proliferated and secreted IFN-gamma and IL-6 in response to Tat. Finally, CTLs were also elicited and they recognized an epitope localized within aa 11-40 of Tat.     

Cholera toxin impairs the differentiation of monocytes into dendritic cells, inducing professional antigen-presenting myeloid cells

Cholera toxin (CT) is a potent adjuvant for mucosal vaccination; however, its mechanism of action has not been clarified completely. It is well established that peripheral monocytes differentiate into dendritic cells (DCs) both in vitro and in vivo and that monocytes are the in vivo precursors of mucosal CD103⁻ proinflammatory DCs. In this study, we asked whether CT had any effects on the differentiation of monocytes into DCs. We found that CT-treated monocytes, in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), failed to differentiate into classical DCs (CD14^low CD1a^high) and acquired a macrophage-like phenotype (CD14^high CD1a^low). Cells differentiated in the presence of CT expressed high levels of major histocompatibility complex class I (MHC-I) and MHC-II and CD80 and CD86 costimulatory molecules and produced larger amounts of IL-1β, IL-6, and IL-10 but smaller amounts of tumor necrosis factor alpha (TNF-α) and IL-12 than did monocytes differentiated into DCs in the absence of CT. The enzymatic activity of CT was found to be important for the skewing of monocytes toward a macrophage-like phenotype (Ma-DCs) with enhanced antigen-presenting functions. Indeed, treatment of monocytes with scalar doses of forskolin (FSK), an activator of adenylate cyclase, induced them to differentiate in a dose-dependent manner into a population with phenotype and functions similar to those found after CT treatment. Monocytes differentiated in the presence of CT induced the differentiation of naïve T lymphocytes toward a Th2 phenotype. Interestingly, we found that CT interferes with the differentiation of monocytes into DCs in vivo and promotes the induction of activated antigen-presenting cells (APCs) following systemic immunization.Adjuvant design has been mainly empirical, and the mechanism of action of the most efficacious molecules has remained obscure. It is important to investigate the mechanisms of action of already known adjuvants to facilitate the design of new effective molecules with high potency and decreased side effects. The bacterial enterotoxin cholera toxin (CT) from Vibrio cholerae is extraordinarily effective as a mucosal and systemic adjuvant, yet its capacity to amplify the immune response has not been completely clarified (12, 24, 25). It is a holotoxin, composed of an enzymatically active A subunit, noncovalently linked to a pentameric B subunit, which binds to the ganglioside GM1 on host cell membranes. Once internalized, the A subunit ADP-ribosylates the α subunit of the GTP-binding regulatory protein Gs, thereby inducing permanent adenylate cyclase activation, resulting in an increase of intracellular cyclic AMP (cAMP) (55). The mechanism of the adjuvanticity of CT may be a complex phenomenon resulting from the interaction of the toxin with different cell types present in the architecture of the mucosa. Dissecting the effect of CT on different cells types could help in understanding the contribution of each to its adjuvant activity.Given the crucial role of dendritic cells (DCs) and other professional antigen-presenting cells (APCs) (monocytes/macrophages and B cells) in the induction of adaptive immunity (26), the potentiation of APC function can be a major aspect of adjuvant action. Cholera toxin upregulates expression of the B7-2 (CD86) costimulatory molecule and stimulates antigen presentation through enhancement of major histocompatibility complex class II (MHC-II) expression and interleukin 1β (IL-1β) production (7, 11, 37, 54). CT also interacts with lymphocytes and promotes B cell isotype-switch differentiation toward IgG1 and IgA in mice (2, 54) and enhances the antigen-presenting function of human B cells (38). We and others showed that CT, by inducing maturation of human DCs, polarizes a mixed Th1/Th2 T cell response, with a strong bias toward Th2 cells (15, 16).The capacity of CT to interact with DCs or monocytes as innate immune cells or as precursors of DCs may represent a crucial step for its adjuvant mechanism. DCs represent heterogeneous populations that comprise distinct subtypes that vary in hematopoietic origin, life cycle, and function (4, 52). It is well established that peripheral monocytes differentiate into DCs both in vitro and in vivo (19, 20, 43-45, 48) and that monocytes are the in vivo precursors of mucosal CD103⁻ proinflammatory DCs (5, 27, 58). More important, the role of monocytes as precursors of DCs in mediating the in vivo adjuvant effects has been described (29, 60). Therefore, in this study, we asked whether CT affected the differentiation of monocytes into DCs. By using human DCs generated from monocytes (48), we found that CT interferes with the differentiation of monocytes into DCs, giving rise to a distinct population (Ma-DCs), which displays an activated macrophage-like phenotype, induces a strong allogeneic and antigen-specific response, and promotes the polarization of naive CD4⁺ T lymphocytes toward a Th2 profile. Interestingly, we found that CT interferes with the differentiation of monocytes into DCs in vivo and promotes the induction of activated antigen-presenting cells following systemic immunization.     

Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin,but Not Their Nontoxic Counterparts,Improve the Antigen-Presenting Cell Function of Human B Lymphocytes

B lymphocytes play an important role in the immune response induced by mucosal adjuvants. In this study we investigated the in vitro antigen-presenting cell (APC) properties of human B cells upon treatment with cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) and nontoxic counterparts of these toxins, such as the B subunit of CT (CT-B) and the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Furthermore, forskolin (FSK), a direct activator of adenylate cyclase, and cyclic AMP (cAMP) analogues were used to investigate the role of the increase in intracellular cAMP caused by the A subunit of CT and LT. B lymphocytes were cultured with adjuvants and polyclonal stimuli necessary for activation of B cells in the absence of CD4 T cells. Data indicated that treatment with CT, LT, FSK, or cAMP analogues, but not treatment with CT-B or LTK63, upregulated surface activation markers on B cells, such as CD86 and HLA-DR, and induced inhibition of the proliferation of B cells at early time points, while it increased cell death in long-term cultures. Importantly, B cells treated with CT, LT, or FSK were able to induce pronounced proliferation of both CD4⁺ and CD8⁺ allogeneic T cells compared with untreated B cells and B cells treated with CT-B and LTK63. Finally, only treatment with toxins or FSK induced antigen-specific T-cell proliferation in Mycobacterium tuberculosis purified protein derivative or tetanus toxoid responder donors. Taken together, these results indicated that the in vitro effects of CT and LT on human B cells are mediated by cAMP.The development of effective mucosal vaccines has been hindered by the lack of useful adjuvants and our limited knowledge of their modes of action. Cholera toxin (CT) from Vibrio cholerae and Escherichia coli heat-labile enterotoxin (LT) are potent immunological adjuvants, as indicated by mouse vaccine studies, although their mechanisms of action are not fully understood. These toxins are holotoxins composed of an enzymatically active A subunit that is noncovalently linked to a pentamer of B subunits binding a variety of galactose-containing molecules present in the plasma membranes of eukaryotic cells. CT binds mostly to the ganglioside GM1, which is believed to be the major toxin receptor, whereas LT binds not only to GM1 but also to other glycosphingolipids. Once internalized, the A subunit ADP ribosylates the α subunit of the GTP-binding regulatory protein Gs, thereby inducing permanent adenylate cyclase activation, resulting in an increase in the level of intracellular cyclic AMP (cAMP) (reviewed in reference 34).The potentiation of antigen-presenting cell (APC) function is a major aspect of adjuvant action, and it has been shown that CT and LT induce maturation of both murine dendritic cells (DC) (26, 36) and human DC (5, 14, 15). Several studies demonstrated the ability of these toxins to promote B-cell isotype switch differentiation in mice (19, 27) and upregulation of activation markers in both murine and human B cells (2-4). While these toxins are potent adjuvants, their toxicity makes them unsuitable for human use. For this reason, a number of investigators have tried to develop nontoxic derivatives of CT and LT that retain adjuvanticity either by removing the A domain or by rendering it enzymatically inactive by site-directed mutagenesis (34). Although the current data suggest that the enzymatic activity of CT and LT holotoxins is responsible for the most potent adjuvant activity, a number of reports proposed that there are multiple immune modulating pathways that are triggered by CT and LT, including mechanisms independent of ADP ribosyltransferase activity (11, 13, 30, 33, 42). Numerous studies have suggested that engagement of the ganglioside GM1, the major receptor for CT and LT, is required for the ability of these molecules to modulate immune responses (22, 31). Recently, workers demonstrated that in the absence of the toxic A subunit, the B subunit of CT (CT-B) induces intracellular signaling associated with the in vitro activation of murine B cells and macrophages (37).The majority of these studies have been performed with murine cells and have confirmed the in vivo adjuvanticity of nontoxic compounds, such as CT-B and LTK63, a mutant of LT lacking the ADP ribosyltransferase enzymatic activity, when they were mucosally delivered into animals, even if the immune responses observed in the in vivo studies were usually weaker than those induced by the wild-type toxins (6, 11, 20, 36, 40, 41). In order to develop a mucosal adjuvant for human vaccine, the mechanism(s) of action of potential nontoxic adjuvants should be investigated in vitro by using human APC. It has been shown that the B-cell antigen-presenting functions may be important for the induction of optimal vaccine-induced responses (10, 35). Moreover, B cells are present in mucosa-associated lymphoid tissues (8), and their function in these sites is related not only to immunoglobulin (Ig) production but also to their antigen-presenting properties (24). To elucidate the mechanisms by which enterotoxins modulate antigen-presenting properties, we decided to carry out a comprehensive and comparative analysis of the effects of the toxins and their nontoxic derivatives on the APC function of human B cells. Here we present evidence that CT and LT, as well as forskolin (FSK) and cAMP analogues, but not CT-B and LTK63, increase the activation of human B cells and induce improvement in their APC capability, indicating that the presence of the enzymatic subunit is critical for their adjuvanticity.     

A modified microcomplement fixation test for antigen or antibody determination in soluble immune complexes, using gel filtration

A Modified Microcomplement Fixation Test (MMFT), useful for detecting and quantifying soluble immune complexes (ICs) and their components in chromatographic separations, is described. This method is based on the addition of excess specific antibody to the ICs against any of their components in order to increase the ICs' anticomplementarity. The concentration of ICs is expressed as the sample dilution which fixes 50% of the added Complement. The MMFT was applied to profiles of ICs obtained in vivo and in vitro. MMFT allows great sensitivity, good reproductibility and the detection of noncomplement fixing ICs without any interference of free antigen (Ag) or free antibody (Ab).     

Carpals and epiphyses of radius and ulna as age indicators using longitudinal data: a Bayesian approach

International Journal of Legal Medicine - The aim of this study is to develop a new formula for age estimation in a longitudinal study of a sample from the radiological collection of wrist bones of...     

Effects of the adjuvant cholera toxin on dendritic cells: stimulatory and inhibitory signals that result in the amplification of immune responses

Cholera toxin (CT) is a potent mucosal adjuvant. When administered through the mucosal route CT amplifies B and T lymphocyte responses to co-administered antigens. Since the discovery of CT as a mucosal adjuvant, other bacterial enterotoxins have been found to have this property. These molecules or their detoxified derivatives are all important for the development of mucosal vaccines for human use, and it is thus necessary to understand their mechanism of action. CT has immunomodulatory effects on different cell types, however, the interaction of CT with dendritic cells (DCs), which have a primary role in the priming of immune responses, may be crucial for its adjuvant activity.