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- Terrinoni, Manuela
(författare)
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Novel approaches to mucosal vaccine development Strategies in vaccine antigen production, construction of a novel mucosal adjuvant and studies of its mode of action
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Terrinoni, Manuela, et al.
(författare)
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Proteomic analysis of cholera toxin adjuvant-stimulated human monocytes identifies Thrombospondin-1 and Integrin-beta 1 as strongly upregulated molecules involved in adjuvant activity
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Terrinoni, Manuela, et al.
(författare)
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Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NF kappa B Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT
- 2019
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- 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 NF kappa B, 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, NF kappa B-/- 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-1 beta, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NF kappa B activation reporter system, we show that CT induced NF kappa B signaling in human monocytes, and that inhibition of the cyclic AMP-protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NF kappa B. 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 NF kappa B signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NF kappa B 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 NF kappa B signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT.
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| 4. |
- Terrinoni, Manuela, et al.
(författare)
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Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NFκB Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT
- 2019
-
Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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