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Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier.
Burggraaf, MJ, Ates, LS, Speer, A, van der Kuij, K, Kuijl, C, Bitter, W
Microbial cell factories. 2019;(1):44
Abstract
BACKGROUND Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. RESULTS We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. CONCLUSION We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.
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The human CIB1-EVER1-EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses.
de Jong, SJ, Créquer, A, Matos, I, Hum, D, Gunasekharan, V, Lorenzo, L, Jabot-Hanin, F, Imahorn, E, Arias, AA, Vahidnezhad, H, et al
The Journal of experimental medicine. 2018;(9):2289-2310
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Abstract
Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of TMC6 (encoding EVER1) or TMC8 (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human β-papillomaviruses (β-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the CIB1 gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to β-HPVs of EV patients.
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The Tumor Antigen NY-ESO-1 Mediates Direct Recognition of Melanoma Cells by CD4+ T Cells after Intercellular Antigen Transfer.
Fonteneau, JF, Brilot, F, Münz, C, Gannagé, M
Journal of immunology (Baltimore, Md. : 1950). 2016;(1):64-71
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Abstract
NY-ESO-1-specific CD4(+) T cells are of interest for immune therapy against tumors, because it has been shown that their transfer into a patient with melanoma resulted in tumor regression. Therefore, we investigated how NY-ESO-1 is processed onto MHC class II molecules for direct CD4(+) T cell recognition of melanoma cells. We could rule out proteasome and autophagy-dependent endogenous Ag processing for MHC class II presentation. In contrast, intercellular Ag transfer, followed by classical MHC class II Ag processing via endocytosis, sensitized neighboring melanoma cells for CD4(+) T cell recognition. However, macroautophagy targeting of NY-ESO-1 enhanced MHC class II presentation. Therefore, both elevated NY-ESO-1 release and macroautophagy targeting could improve melanoma cell recognition by CD4(+) T cells and should be explored during immunotherapy of melanoma.
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Regulation of the trafficking and antiviral activity of IFITM3 by post-translational modifications.
Chesarino, NM, McMichael, TM, Yount, JS
Future microbiology. 2014;(10):1151-63
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Abstract
IFITM3 restricts cellular infection by multiple important viral pathogens, and is particularly critical for the innate immune response against influenza virus. Expression of IFITM3 expands acidic endolysosomal compartments and prevents fusion of endocytosed viruses, leading to their degradation. This small, 133 amino acid, antiviral protein is controlled by at least four distinct post-translational modifications. Positive regulation of IFITM3 antiviral activity is provided by S-palmitoylation, while negative regulatory mechanisms include lysine ubiquitination, lysine methylation and tyrosine phosphorylation. Herein, we describe specific insights into IFITM3 trafficking and activity that were provided by studies of IFITM3 post-translational modifications, and discuss evidence suggesting that IFITM3 adopts multiple membrane topologies involving at least one intramembrane domain in its antivirally active conformation.
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Flavonoid-membrane interactions: involvement of flavonoid-metal complexes in raft signaling.
Tarahovsky, YS, Kim, YA, Yagolnik, EA, Muzafarov, EN
Biochimica et biophysica acta. 2014;(5):1235-46
Abstract
Flavonoids are polyphenolic compounds produced by plants and delivered to the human body through food. Although the epidemiological analyses of large human populations did not reveal a simple correlation between flavonoid consumption and health, laboratory investigations and clinical trials clearly demonstrate the effectiveness of flavonoids in the prevention of cardiovascular, carcinogenic, neurodegenerative and immune diseases, as well as other diseases. At present, the abilities of flavonoids in the regulation of cell metabolism, gene expression, and protection against oxidative stress are well-known, although certain biophysical aspects of their functioning are not yet clear. Most flavonoids are poorly soluble in water and, similar to lipophilic compounds, have a tendency to accumulate in biological membranes, particularly in lipid rafts, where they can interact with different receptors and signal transducers and influence their functioning through modulation of the lipid-phase behavior. In this study, we discuss the enhancement in the lipophilicity and antioxidative activity of flavonoids after their complexation with transient metal cations. We hypothesize that flavonoid-metal complexes are involved in the formation of molecular assemblies due to the facilitation of membrane adhesion and fusion, protein-protein and protein-membrane binding, and other processes responsible for the regulation of cell metabolism and protection against environmental hazards.
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Galectin-9 signaling through TIM-3 is involved in neutrophil-mediated Gram-negative bacterial killing: an effect abrogated within the cystic fibrosis lung.
Vega-Carrascal, I, Bergin, DA, McElvaney, OJ, McCarthy, C, Banville, N, Pohl, K, Hirashima, M, Kuchroo, VK, Reeves, EP, McElvaney, NG
Journal of immunology (Baltimore, Md. : 1950). 2014;(5):2418-31
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Abstract
The T cell Ig and mucin domain-containing molecule (TIM) family of receptors have emerged as potential therapeutic targets to correct abnormal immune function in chronic inflammatory conditions. TIM-3 serves as a functional receptor in structural cells of the airways and via the ligand galectin-9 (Gal-9) can modulate the inflammatory response. The aim of this study was to investigate TIM-3 expression and function in neutrophils, focusing on its potential role in cystic fibrosis (CF) lung disease. Results revealed that TIM-3 mRNA and protein expression values of circulating neutrophils were equal between healthy controls (n = 20) and people with CF (n = 26). TIM-3 was detected on resting neutrophil membranes by FACS analysis, and expression levels significantly increased post IL-8 or TNF-α exposure (p < 0.05). Our data suggest a novel role for TIM-3/Gal-9 signaling involving modulation of cytosolic calcium levels. Via TIM-3 interaction, Gal-9 induced neutrophil degranulation and primed the cell for enhanced NADPH oxidase activity. Killing of Pseudomonas aeruginosa was significantly increased upon bacterial opsonization with Gal-9 (p < 0.05), an effect abrogated by blockade of TIM-3 receptors. This mechanism appeared to be Gram-negative bacteria specific and mediated via Gal-9/ LPS binding. Additionally, we have demonstrated that neutrophil TIM-3/Gal-9 signaling is perturbed in the CF airways due to proteolytic degradation of the receptor. In conclusion, results suggest a novel neutrophil defect potentially contributing to the defective bacterial clearance observed in the CF airways and suggest that manipulation of the TIM-3 signaling pathway may be of therapeutic value in CF, preferably in conjunction with antiprotease treatment.
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Dose-dependent effects of NY-ESO-1 protein vaccine complexed with cholesteryl pullulan (CHP-NY-ESO-1) on immune responses and survival benefits of esophageal cancer patients.
Kageyama, S, Wada, H, Muro, K, Niwa, Y, Ueda, S, Miyata, H, Takiguchi, S, Sugino, SH, Miyahara, Y, Ikeda, H, et al
Journal of translational medicine. 2013;:246
Abstract
BACKGROUND Cholesteryl pullulan (CHP) is a novel antigen delivery system for cancer vaccines. This study evaluated the safety, immune responses and clinical outcomes of patients who received the CHP-NY-ESO-1 complex vaccine, Drug code: IMF-001. METHODS Patients with advanced/metastatic esophageal cancer were enrolled and subcutaneously vaccinated with either 100 μg or 200 μg of NY-ESO-1 protein complexed with CHP. The primary endpoints were safety and humoral immune responses, and the secondary endpoint was clinical efficacy. RESULTS A total of 25 patients were enrolled. Thirteen and twelve patients were repeatedly vaccinated with 100 μg or 200 μg of CHP-NY-ESO-1 with a median of 8 or 9.5 doses, respectively. No serious adverse events related to the vaccine were observed. Three out of 13 patients in the 100-μg cohort and 7 out of 12 patients in the 200-μg cohort were positive for anti-NY-ESO-1 antibodies at baseline. In the 100-μg cohort, an antibody response was observed in 5 out of 10 pre-antibody-negatives patients, and the antibody levels were augmented in 2 pre-antibody-positive patients after vaccination. In the 200-μg cohort, all 5 pre-antibody-negative patients became seropositive, and the antibody level was amplified in all 7 pre-antibody-positive patients. No tumor shrinkage was observed. The patients who received 200 μg of CHP-NY-ESO-1 survived longer than patients receiving 100 μg of CHP-NY-ESO-1, even those who exhibited unresponsiveness to previous therapies or had higher tumor burdens. CONCLUSIONS The safety and immunogenicity of CHP-NY-ESO-1 vaccine were confirmed. The 200 μg dose more efficiently induced immune responses and suggested better survival benefits. (Clinical trial registration number NCT01003808).
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Immunocapture and identification of cell membrane protein antigenic targets of serum autoantibodies.
Littleton, E, Dreger, M, Palace, J, Vincent, A
Molecular & cellular proteomics : MCP. 2009;(7):1688-96
Abstract
There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies. To identify cell surface antigens, we developed a "conformational membrane antigen isolation and identification" strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and immune complexes are precipitated. Third, the bound surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific tyrosine receptor kinase (MuSK) and sera or plasmas from patients with a subtype of the autoimmune disease myasthenia gravis in which patients have autoantibodies against MuSK. MuSK was robustly detected as the only membrane protein in immunoprecipitates from all three patient samples tested and not from the three MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from cell extracts. The conformational membrane antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The approach would also be very suitable for the analysis of human or experimental antitumor responses.
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A randomized and controlled Phase 1 study of the safety and immunogenicity of the AMA1-C1/Alhydrogel + CPG 7909 vaccine for Plasmodium falciparum malaria in semi-immune Malian adults.
Sagara, I, Ellis, RD, Dicko, A, Niambele, MB, Kamate, B, Guindo, O, Sissoko, MS, Fay, MP, Guindo, MA, Kante, O, et al
Vaccine. 2009;(52):7292-8
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Abstract
A double blind, randomized and controlled Phase 1 clinical trial was conducted to assess the safety and immunogenicity in malaria-exposed adults of the Plasmodium falciparum blood stage vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1)/Alhydrogel with and without the novel adjuvant CPG 7909. Participants were healthy adults 18-45 years old living in the village of Donéguébougou, Mali. A total of 24 participants received 2 doses one month apart of either 80 microg AMA1-C1/Alhydrogel or 80 microg AMA1-C1/Alhydrogel + 564 microg CPG 7909. The study started in October 2007 and completed follow up in May 2008. Both vaccines were well tolerated, with only mild local adverse events and no systemic adverse events judged related to vaccination. The difference in antibody responses were over 2-fold higher in the group receiving CPG 7909 for all time points after second vaccination and the differences are statistically significant (all p<0.05). This is the first use of the novel adjuvant CPG 7909 in a malaria-exposed population.
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Dectin-1 is required for human dendritic cells to initiate immune response to Candida albicans through Syk activation.
Skrzypek, F, Cenci, E, Pietrella, D, Rachini, A, Bistoni, F, Vecchiarelli, A
Microbes and infection. 2009;(6-7):661-70
Abstract
Dectin-1 is a pattern recognition receptor found on monocytes and dendritic cells (DC) able to recognize beta-1,3 and beta-1,6 glucans. It is thought to act via the spleen tyrosine kinase (Syk) to initiate immune response against infectious agents such as Candida albicans, one of the leading causes of invasive fungal disease in immunocompromised individuals. This study addresses the importance of this receptor in the context of human DC response to C. albicans. Upon blockage of Dectin-1, immature DC are less able than untreated cells to bind, phagocytose, and kill C. albicans via oxidative burst. In fact, a consistent decrease in superoxide anion, but not nitric oxide production, was manifested when the Syk pathway was inhibited. C. albicans-induced cytokine production via Dectin-1 recognition is mediated by the Syk activation pathway. Indeed, specific Syk inhibition significantly suppressed the production of IL-12, IL-6, and TNF-alpha. Finally, we observed that Dectin-1 engagement was also involved in DC maturation and subsequent lymphocyte activation. Collectively, these findings identify Dectin-1 as a key receptor influencing critical biological functions of DC in response to C. albicans leading to T cells response alteration. These effects are largely, though not completely, mediated by Syk activation.