Maria Teresa De Magistris

Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming

Cholera toxin (CT) is a potent mucosal adjuvant that amplifies B and T cell responses to mucosally co‐administered antigens, stimulating predominant Th2‐type responses. However, little is known about the mechanism of adjuvanticity of CT and on the influence this toxin may have on Th2 cell development during the priming of an immune response. We analyzed the effect of CT on dendritic cells (DC), which are responsible for the priming of immune responses at the systemic as well as at the mucosal level. We found that CT induces phenotypic and functional maturation of blood monocyte‐derived DC. Indeed, CT‐treated DC up‐regulate expression of HLA‐DR molecules, B7.1 and B7.2 co‐stimulatory molecules, and are able to prime naive CD4+CD45RA+ T cells in vitro, driving their polarization towards the Th2 phenotype. Furthermore, CT‐matured DC express functional chemokine receptors CCR7 and CXCR4 which may render them responsive to migratory stimuli towards secondary lymphoid organs. Interestingly, the maturation program induced by CT is unique since CT does not induce but rather inhibits cytokine (IL‐12p70 and TNF‐α) and chemokine (RANTES, MIP‐1α and MIP‐1β) secretion by lipopolysaccharide‐ or CD40 ligand‐activated DC. Our results help to elucidate the mechanism of action of CT as an adjuvant and highlight a new stimulus of bacterial origin that promotes maturation of DC.

Rationally designed strings of promiscuous CD4+ T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines

The age‐related and T cell‐independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long‐lasting T helper cell immunity. Herewe describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4+ T cell epitopes from various pathogen‐derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti‐Hib antibody response in mice. In the case of the N19‐Hib conjugate, this response is at least as good as that observed with CRM197‐Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

Phase I clinical trial of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin combined with FHA and 69 kDa

An acellular pertussis vaccine composed of genetically detoxified pertussis toxin (PT-9K/129G), filamentous haemagglutinin (FHA) and pertactin (69 kDa protein) was evaluated in adult volunteers, in double blind, versus placebo. No fever was reported in either group. Mild local reactions were reported after injection of both vaccine and placebo. After the first dose a marked increase in antibodies to PT, FHA and 69 kDa protein was seen in vaccinated subjects with the exception of one who responded well to PT and FHA but did not show a humoral response to the 69 kDa protein. All vaccinees acquired cellular immunity against the three antigens. No significant variation was observed in the humoral or cellular responses after the second dose.

Cyclic Adenosine 5′-Monophosphate and Calcium Induce CD152 (CTLA-4) Up-Regulation in Resting CD4+ T Lymphocytes

The CTLA-4 (CD152) molecule is up-regulated upon T cell activation and proliferation, and plays a critical role in the inhibition of immune responses. We show in this study that cAMP induces up-regulation of CD152 in human CD4+ T lymphocytes. This effect occurs in the absence of the up-regulation of CD69 and CD25 activation markers and T cell proliferation. In addition, we found that the Ca2+ ionophore ionomycin also up-regulates CD152, and that the combination of a cAMP analog or cAMP inducers with ionomycin further enhances the expression of CD152 in resting CD4+ T lymphocytes. However, cyclosporin A, which inhibits Ca2+/calcineurin signaling pathway, fully prevented the ionomycin- but not the cAMP-induced up-regulation of CD152. The effects of cAMP and ionomycin involve increase of both CD152 mRNA transcripts, coding for the membrane and the soluble forms of CD152. Furthermore, we show that CD152 molecules are translocated to the membrane and are functional, as their engagement by specific mAbs prevented NF-κB activation by anti-CD3/CD28 stimulation. These findings demonstrate that at least two novel signal pathways regulate CTLA-4 gene expression and CD152 molecule up-regulation in human CD4+ T lymphocytes, in the absence of full T cell activation.

Maturation of human dendritic cells induced by the adjuvant cholera toxin: role of cAMP on chemokine receptor expression

Cholera toxin (CT) is a very effective adjuvant for mucosal vaccination. It binds to cells through its B subunit and induces intracellular increase of cAMP through the A subunit. We previously showed that CT induces maturation of human dendritic cells (DCs) and this may account for its adjuvant property. Here, we investigated the role of the A subunit on DCs maturation by using forskolin, a cAMP inducer. The results show that although cAMP does not stimulate full maturation of DCs it induces upregulation of the chemokine receptors CXCR4 and CCR7.

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.

Persistence of mucosal and systemic immune responses following sublingual immunization

The development of mucosal vaccines for prevention of infectious diseases caused by pathogens entering through the mucosal surfaces is an important and challenging objective. To this purpose, we evaluated the efficacy and durability of immune response induced by sublingual immunization with tetanus toxoid (TT) as an antigen in the presence of mucosal adjuvants, such as E. coli Heat-Labile enterotoxin (LT) or the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Both serum anti-TT IgG and mucosal anti-TT IgA antibodies reached a peak after four immunizations and decreased over time, maintaining detectable titers up to 4 months after the last immunization. Similarly, antigen-specific antibody secreting cells in bone marrow and TT-specific CD4+ and CD8+ T cells in draining lymph nodes and spleen were present up to 4 months from the last immunization. Overall, LT-treated mice showed significantly higher responses compared to LTK63 immunized mice. The efficacy and persistence of the immune response induced by sublingual immunization with different adjuvants strongly suggest that this route represents an appealing and promising alternative to the other mucosal routes of vaccine delivery.

Human CD4+ T lymphocytes with increased intracellular cAMP levels exert regulatory functions by releasing extracellular cAMP

We have previously shown that cholera toxin (CT) and other cAMP‐elevating agents induce up‐regulation of the inhibitory molecule CTLA‐4 on human resting T lymphocytes. In this study, we evaluated the function of these cells. We found that purified human CD4+ T lymphocytes pretreated with CT were able to inhibit proliferation of autologous PBMC in a dose‐dependent manner. It is interesting that this phenomenon was not mediated by inhibitory cytokines such as IL‐10, IL‐4, or TGF‐β but was in part caused by the release of extracellular cAMP by the CD4+ T lymphocytes. Purified CD4+ T cells pretreated with forskolin, a transient cAMP inducer, or with dibutyryl cAMP, an analog of cAMP, did not exert suppressive functions, suggesting that a sustained production of cAMP, such as that induced by CT, was required to identify a novel regulatory function mediated by CD4+ T cells. Our results show that CD4+ T lymphocytes can exert regulatory functions through the release of extracellular cAMP and that the cyclic nucleotide acts as a primary messenger, which could play a biological role in the modulation of immune responses.

Progress towards the development of new vaccines against whooping cough

Acellular vaccines against whooping cough are in the final stage of clinical testing and are likely to become available for mass immunization in the near future. Over a dozen vaccines of similar composition have been developed by vaccine companies and research laboratories; all of them contain a detoxified form of pertussis toxin (PT) that may be present alone or combined with one or more other non-toxic proteins, such as filamentous haemagglutinin (FHA), pertactin (69 kDa), and the agglutinogens (AGG). Most of the vaccines contain a PT that has been inactivated by chemical treatment, a process that reduces the immunogenicity of the molecule and may not completely eliminate the risk of reversion to toxicity. To avoid these problems, we have constructed by genetic manipulation a mutant of Bordetella pertussis that produces a non-toxic form of PT. This molecule (PT-9K/129G) contains two amino acid substitutions in the S1 subunit (Arg9-->Lys and Glu129-->Gly) which abolish the enzymatic activity of the S1 subunit and all the toxic properties of PT, without changing the immunological properties of the wild-type toxin. Following extensive preclinical studies, which have shown that PT-9K/129G is safe and more antigenic than the toxin treated with chemical agents, this molecule was tested for safety and immunogenicity in adult volunteers, 18-month-old children and 2-month-old infants. The molecule has been tested alone, combined with FHA and pertactin and also combined with diphtheria and tetanus toxoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Human monocytes respond to extracellular cAMP through A2A and A2B adenosine receptors

In this study, we test the hypothesis that cAMP, acting as an extracellular mediator, affects the physiology and function of human myeloid cells. The cAMP is a second messenger recognized as a universal regulator of several cellular functions in different organisms. Many studies have shown that extracellular cAMP exerts regulatory functions, acting as first mediator in multiple tissues. However, the impact of extracellular cAMP on cells of the immune system has not been fully investigated. We found that human monocytes exposed to extracellular cAMP exhibit higher expression of CD14 and lower amount of MHC class I and class II molecules. When cAMP‐treated monocytes are exposed to proinflammatory stimuli, they exhibit an increased production of IL‐6 and IL‐10 and a lower amount of TNF‐α and IL‐12 compared with control cells, resembling the features of the alternative‐activated macrophages or M2 macrophages. In addition, we show that extracellular cAMP affects monocyte differentiation into DCs, promoting the induction of cells displaying an activated, macrophage‐like phenotype with reduced capacity of polarized, naive CD4+ T cells into IFN‐γ‐producing lymphocytes compared with control cells. The effects of extracellular cAMP on monocytes are mediated by CD73 ecto‐5′‐nucleotidase and A2A and A2B adenosine receptors, as selective antagonists could reverse its effects. Of note, the expression of CD73 molecules has been found on the membrane of a small population of CD14+CD16+ monocytes. These findings suggest that an extracellular cAMP‐adenosine pathway is active in cells of the immune systems.