Novel approaches to mucosal vaccine development Strategies in vaccine antigen production, construction of a novel mucosal adjuvant and studies of its mode of action

Although most infections begin at a mucosal surface and may be prevented by effective vaccine stimulation of the local mucosal immune system, there are so far only a few mucosal vaccines available for human use. This thesis spans several areas that are important for future development of mucosal vaccines. Future vaccine development will depend in part on the efficient production of recombinant antigens produced in bacterial expression systems. To avoid current problems with the use of antibiotics to maintain expression plasmids, an E. coli strain capable of producing recombinant proteins using vectors maintained without the need antibiotic was generated. The method is based on deletion of the essential lgt gene encoding a (pro)lipoprotein glyceryl transferase and complementing it with an expression vector carrying the non-homologous lgt gene from V. cholerae. A similar V. cholerae lgt-deleted strain was also constructed using the E. coli lgt gene for complementation. The strains had similar growth and production characteristics as their wild-type counterparts but maintained their expression plasmids without the need for antibiotics. The system was used to express two recombinant vaccine proteins, cholera toxin B subunit and a fusion protein for vaccination against atherosclerosis. In the development of mucosal vaccines, it is often important to enhance immune responses using adjuvants, since most mucosally administered antigens are poorly immunogenic. Cholera toxin (CT) is the most powerful mucosal adjuvant known but is too toxic for human use. A mutated CT derivative (mmCT) was constructed and expressed in an engineered strain of V. cholerae. mmCT induced 1000 times less cAMP than native CT in a mouse thymocyte toxicity assay, was non-toxic in an infant mouse model and yet retained similar adjuvant properties as native CT. We suggest that mmCT is a promising candidate for use in future mucosal vaccines. The mode of adjuvant action of mmCT and native CT was investigated using human and mouse antigen-presenting cells, which are primary target cells for adjuvants. Both molecules were found to activate cyclic AMP/protein kinase A-dependent canonical NF-kB signaling associated with inflammasome activation. The activation of these pathways was found to induce expression of two immunomodulatory proteins, THSB1 and ITGB1, as well as increased expression and activation of IL-1β, a cytokine which has been shown to play an important role for the adjuvant action of CT and mmCT

Proteomic analysis of cholera toxin adjuvant-stimulated human monocytes identifies Thrombospondin-1 and Integrin-beta 1 as strongly upregulated molecules involved in adjuvant activity

Cholera Toxin (CT) as well as its related non-toxic mmCT and dmLT mutant proteins have been shown to be potent adjuvants for mucosally administered vaccines. Their adjuvant activity involves activation of cAMP/protein kinase A (PKA) signaling and inflammasome/IL-1 beta pathways in antigen presenting cells (APC). To get a further understanding of the signal transduction and downstream pathways activated in APCs by this group of adjuvants we have, employing quantitative proteomic analytic tools, investigated human monocytes at various time points after treatment with CT. We report the activation of three main biological pathways among upregulated proteins, peaking at 16 hours of CT treatment: cellular organization, metabolism, and immune response. Specifically, in the further analyzed immune response pathway we note a strong upregulation of thrombospondin 1 (THBS1) and integrin beta 1 (ITGB1) in response to CT as well as to mmCT and dmLT, mediated via cAMP/PKA and NFKB signaling. Importantly, inhibition in vitro of THSB1 and ITGB1 in monocytes or primary dendritic cells using siRNA abrogated the ability of the treated APCs to promote an adjuvant-stimulated Th17 cell response when co-cultured with peripheral blood lymphocytes indicating the involvement of these molecules in the adjuvant action on APCs by CT, mmCT and dmLT

Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NFκB Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT

Cholera toxin (CT) is widely used as an effective adjuvant in experimental immunology for inducing mucosal immune responses; yet its mechanisms of adjuvant action remain incompletely defined. Here, we demonstrate that mice lacking NFκB, compared to wild-type (WT) mice, had a 90% reduction in their systemic and mucosal immune responses to oral immunization with a model protein antigen [Ovalbumin (OVA)] given together with CT. Further, NFκB−/− mouse dendritic cells (DCs) stimulated in vitro with CT showed reduced expression of MHCII and co-stimulatory molecules, such as CD80 and CD86, as well as of IL-1β, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NFκB activation reporter system, we show that CT induced NFκB signaling in human monocytes, and that inhibition of the cyclic AMP—protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NFκB. In a human monocyte-CD4+ T cell co-culture system we further show that the strong Th17 response induced by CT treatment of monocytes was abolished by blocking the classical but not the alternative NFκB signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NFκB pathway signaling in antigen-presenting cells (APCs) by CT is important for CT's adjuvant enhancement of Th17 responses. Similar findings were obtained using the almost completely detoxified mmCT mutant protein as adjuvant. Altogether, our results demonstrate that activation of the classical NFκB signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT

Human whole-cell / B-subunit oral cholera vaccines Duochol and Dukoral induce antibody responses in a porcine model

Oral cholera vaccine (OCV) is a critical tool in the WHO’s global program for cholera control and elimination. Before testing new vaccines in humans preclinical testing in animals is performed. Pigs have some advantages over mice in that their anatomical size and structure, their immunology, genome, and physiology, are more closer to these of humans, making them a more appropriate model to test an oral vaccination strategy intended to be used in humans than mice are. Pigs can for instance swallow capsule formulations unable to be swallowed by mice. Here a new low-cost, thermostable dry formulation of whole-cell / recombinant B-subunit (CTB) in enterocoated capsules (Duochol® OCV) was compared with the liquid commercial Dukoral® OCV. Both were administered three times with a 3-week interval to groups of 7 pigs. Serum IgG and IgA anti-Inaba and anti-Ogawa LPS as well as anti-CTB antibody responses were determined by ELISA and vibriocidal serum antibody levels against Vibrio cholerae O1 strains Inaba and Ogawa using guinea pig complement. Peripheral blood mononuclear cells were isolated to determine the antibodies in lymphocyte supernatant (ALS) response. Both Duochol and the liquid Dukoral were immunogenic in pigs, resulting in highly significant vibriocidal antibody responses to both Inaba and Ogawa bacteria in 7 out of 7 pigs that had received Duochol and in 6 of the 7 pigs who obtained Dukoral. There was also an immune response to CTB in most of the immunized animals however less impressive than the vibriocidal antibody responses, most likely due to preexisting antitoxin titers in some animals, probably a reflection to natural exposure to enterotoxigenic Escherichia coli. Duochol OCV was safe and showed equivalent immunogenicity as Dukoral. This is to our knowledge the first study ever in pigs to determine the immune response in pigs to any OCV whether liquid or capsule. The authors acknowledge the Welcome Trust for the financial support of both the Duochol vaccine as well as the present study

Characterization of the ganglioside recognition profile of Escherichia coli heat-labile enterotoxin LT-IIc

International audienceThe heat-labile enterotoxins of Escherichia coli and cholera toxin of Vibrio cholerae are related in structure and function. Each of these oligomeric toxins is comprised of one A polypeptide and five B polypeptides. The B-subunits bind to gangliosides, which are followed by uptake into the intoxicated cell and activation of the host’s adenylate cyclase by the A-subunits. There are two antigenically distinct groups of these toxins. Group I includes cholera toxin and type I heat-labile enterotoxin of E. coli; group II contains the type II heat-labile enterotoxins of E. coli. Three variants of type II toxins, designated LT-IIa, LT-IIb and LT-IIc have been described. Earlier studies revealed the crystalline structure of LT-IIb. Herein the carbohydrate binding specificity of LT-IIc B-subunits was investigated by glycosphingolipid binding studies on thin-layer chromatograms and in microtiter wells. Binding studies using a large variety of glycosphingolipids showed that LT-IIc binds with high affinity to gangliosides with a terminal Neu5Acα3Gal or Neu5Gcα3Gal, e.g. the gangliosides GM3, GD1a and Neu5Acα3-/Neu5Gcα3--neolactotetraosylceramide and Neu5Acα3-/Neu5Gcα3-neolactohexaosylceramide. The crystal structure of LT-IIc B-subunits alone and with bound LSTd/sialyl-lacto-N-neotetraose d pentasaccharide uncovered the molecular basis of the ganglioside recognition. These studies revealed common and unique functional structures of the type II family of heat-labile enterotoxins

Deficiency in Calcium-Binding Protein S100A4 Impairs the Adjuvant Action of Cholera Toxin

The calcium-binding protein S100A4 has been described to promote pathological inflammation in experimental autoimmune and inflammatory disorders and in allergy and to contribute to antigen presentation and antibody response after parenteral immunization with an alum-adjuvanted antigen. In this study, we extend these findings by demonstrating that mice lacking S100A4 have a defective humoral and cellular immune response to mucosal (sublingual) immunization with a model protein antigen [ovalbumin (OVA)] given together with the strong mucosal adjuvant cholera toxin (CT), and that this impairment is due to defective adjuvant-stimulated antigen presentation by antigen-presenting cells. In comparison to wild-type (WT) mice, mice genetically lacking S100A4 had reduced humoral and cellular immune responses after immunization with OVA plus CT, including a complete lack of detectable germinal center reaction. Further, when stimulated in vitro with OVA plus CT, S100A4−/− dendritic cells (DCs) showed impaired responses in several CT-stimulated immune regulatory molecules including the co-stimulatory molecule CD86, inflammasome-associated caspase-1 and IL-1β. Coculture of OVA-specific OT-II T cells with S100A4−/− DCs that had been pulse incubated with OVA plus CT resulted in impaired OT-II T cell proliferation and reduced production of Th1, Th2, and Th17 cytokines compared to similar cocultures with WT DCs. In accordance with these findings, transfection of WT DCs with S100A4-targeting small interfering RNA (siRNA) but not mock-siRNA resulted in significant reductions in the expression of caspase-1 and IL-1β as well as CD86 in response to CT. Importantly, also engraftment of WT DCs into S100A4−/− mice effectively restored the immune response to immunization in the recipients. In conclusion, our results demonstrate that deficiency in S100A4 has a strong impact on the development of both humoral and cellular immunity after mucosal immunization using CT as adjuvant

In Vivo Identification of H3K9me2/H3K79me3 as an Epigenetic Barrier to Carcinogenesis

The highly dynamic nature of chromatin's structure, due to the epigenetic alterations of histones and DNA, controls cellular plasticity and allows the rewiring of the epigenetic landscape required for either cell differentiation or cell (re)programming. To dissect the epigenetic switch enabling the programming of a cancer cell, we carried out wide genome analysis of Histone 3 (H3) modifications during osteogenic differentiation of SH-SY5Y neuroblastoma cells. The most significant modifications concerned H3K27me2/3, H3K9me2, H3K79me1/2, and H3K4me1 that specify the process of healthy adult stem cell differentiation. Next, we translated these findings in vivo, assessing H3K27, H3K9, and H3K79 methylation states in biopsies derived from patients affected by basalioma, head and neck carcinoma, and bladder tumors. Interestingly, we found a drastic decrease in H3K9me2 and H3K79me3 in cancer specimens with respect to their healthy counterparts and also a positive correlation between these two epigenetic flags in all three tumors. Therefore, we suggest that elevated global levels of H3K9me2 and H3K79me3, present in normal differentiated cells but lost in malignancy, may reflect an important epigenetic barrier to tumorigenesis. This suggestion is further corroborated, at least in part, by the deranged expression of the most relevant H3 modifier enzymes, as revealed by bioinformatic analysis. Overall, our study indicates that the simultaneous occurrence of H3K9me2 and H3K79me3 is fundamental to ensure the integrity of differentiated tissues and, thus, their combined evaluation may represent a novel diagnostic marker and potential therapeutic target

B cells treated with CTB-p210 acquire a regulatory phenotype in vitro and reduce atherosclerosis in apolipoprotein E deficient mice

Objective: Intranasal immunization with a fusion protein of the ApoB100-derived peptide p210 and the cholera toxin B subunit (CTB-p210) has previously been shown to induce mucosal tolerance and reduce atherosclerosis development, but the exact mode of action remains to be elucidated. Recent studies have indicated an important role for B cells in mucosal tolerance, in particular by induction of regulatory B (Bregs) and T cells (Tregs). In this study, we aimed to investigate if transfer of B cells pulsed with CTB-p210 can protect against atherosclerosis. Method and results: First, we studied if CTB-p210 can induce Bregs and Tregs in vitro. After pulsing B cells from Apobtm2Sgyldlr−/− or Apoe−/− mice with CTB-p210 for 1 h and co-culturing them with naïve T cells for 48 h, we observed increased expression of membrane bound TGFβ/latency-associated peptide (mTGFβ/LAP) on B cells and an increased proportion of CD25hiFoxP3+ Tregs. Adoptive transfer of B cells pulsed with CTB-p210 into high-fat diet-fed Apoe−/− mice at 8, 10 and 12 weeks of age, reduced the plaque area in the aorta at 20 weeks of age as compared with control-treated (CTB-pOVA treated B cells or PBS) mice. Moreover, mice receiving p210-CTB treated B cells had increased levels of anti-p210 IgG antibodies. Conclusion: Our observations suggest that CTB-p210 pulsed B cells acquire a regulatory phenotype and induce Tregs in vitro. Adoptive transfer of CTB-p210, but not control-treated, B cells into Apoe−/− mice decreased atherosclerosis development

Immune responses against oxidized LDL as possible targets for prevention of atherosclerosis in systemic lupus erythematosus

Patients suffering from systemic lupus erythematosus (SLE) are at increased risk of developing cardiovascular disease (CVD) and traditional therapies including statins provide insufficient protection. Impaired removal of apoptotic material is a common pathogenic mechanism in both SLE and atherosclerosis and is considered to be a key factor in the development of autoimmunity. Since oxidized LDL and apoptotic material bind to the same receptors, we aimed to investigate if targeting the oxidized LDL autoimmunity can affect atherosclerosis in SLE. To investigate the possible role of oxidized LDL autoimmunity in the accelerated atherosclerosis associated with SLE we used a hypercholesterolemic SLE mouse model (B6.lpr.ApoE−/− mice). Promoting LDL tolerance through mucosal immunization with an apolipoprotein B-100 peptide p45 (amino acids 661–680) and cholera toxin B-subunit fusion protein increased regulatory T cells and B cells in mesenteric lymph nodes and reduced plaque development in the aorta by 33%. Treatment with the oxidized LDL-specific antibody Orticumab reduced aortic atherosclerosis by 43%, subvalvular plaque area by 50% and the macrophage content by 31%. The present study provides support for oxLDL as a possible target for prevention of cardiovascular complications in SLE