A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell ResponsesIn Vivo

The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with aKd~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβby TLR4-expressing cells, as well as the production of TNF-αby the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

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Murine Cytomegalovirus Abortively Infects Human Dendritic Cells, Leading to Expression and Presentation of Virally Vectored Genes

Journal of Virology ◽

10.1128/jvi.77.13.7182-7192.2003 ◽

2003 ◽

Vol 77 (13) ◽

pp. 7182-7192 ◽

Cited By ~ 22

Author(s):

Xiuqing Wang ◽

Martin Messerle ◽

Ramil Sapinoro ◽

Kathlyn Santos ◽

Peter K. Hocknell ◽

...

Keyword(s):

Dendritic Cells ◽

Fluorescent Protein ◽

Vaccine Development ◽

Human Monocyte ◽

Surface Proteins ◽

Murine Cytomegalovirus ◽

Specific Class ◽

Green Fluorescent ◽

Human Dendritic Cells ◽

Hiv 1

ABSTRACT Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development.

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Green fluorescent protein expression in dendritic cells enhances their immunogenicity and elicits specific cytotoxic T-cell responses in humans

Experimental Hematology ◽

10.1016/j.exphem.2003.10.014 ◽

2004 ◽

Vol 32 (2) ◽

pp. 210-217 ◽

Cited By ~ 21

Author(s):

F Re

Keyword(s):

Dendritic Cells ◽

Green Fluorescent Protein ◽

T Cell ◽

Protein Expression ◽

Green Fluorescent Protein Expression ◽

Fluorescent Protein ◽

Cytotoxic T Cell ◽

Cell Responses ◽

Cytotoxic T Cell Responses ◽

Green Fluorescent

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Retrovirally Transduced CD34++ Human Cord Blood Cells Generate T Cells Expressing High Levels of the Retroviral Encoded Green Fluorescent Protein Marker In Vitro

Blood ◽

10.1182/blood.v91.2.431.431_431_440 ◽

1998 ◽

Vol 91 (2) ◽

pp. 431-440 ◽

Cited By ~ 2

Author(s):

Bruno Verhasselt ◽

Magda De Smedt ◽

Rita Verhelst ◽

Evelien Naessens ◽

Jean Plum

Keyword(s):

Gene Therapy ◽

Dendritic Cells ◽

Green Fluorescent Protein ◽

T Cell ◽

Gene Transfer ◽

Cord Blood ◽

Fluorescent Protein ◽

Marker Gene ◽

Green Fluorescent

Human umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin−) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin− and CD34++CD38−Lin− UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.

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Retrovirally Transduced CD34++ Human Cord Blood Cells Generate T Cells Expressing High Levels of the Retroviral Encoded Green Fluorescent Protein Marker In Vitro

Blood ◽

10.1182/blood.v91.2.431 ◽

1998 ◽

Vol 91 (2) ◽

pp. 431-440 ◽

Cited By ~ 45

Author(s):

Bruno Verhasselt ◽

Magda De Smedt ◽

Rita Verhelst ◽

Evelien Naessens ◽

Jean Plum

Keyword(s):

Gene Therapy ◽

Dendritic Cells ◽

Green Fluorescent Protein ◽

T Cell ◽

Gene Transfer ◽

Cord Blood ◽

Fluorescent Protein ◽

Marker Gene ◽

Green Fluorescent

Abstract Human umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin−) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin− and CD34++CD38−Lin− UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.

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Protection against Autoimmune Diabetes by Silkworm-Produced GFP-Tagged CTB-Insulin Fusion Protein

Clinical and Developmental Immunology ◽

10.1155/2011/831704 ◽

2011 ◽

Vol 2011 ◽

pp. 1-14 ◽

Cited By ~ 5

Author(s):

Qiaohong Meng ◽

Wenfeng Wang ◽

Xiaowen Shi ◽

Yongfeng Jin ◽

Yaozhou Zhang

Keyword(s):

Fusion Protein ◽

Oral Administration ◽

Fluorescent Protein ◽

Autoimmune Diabetes ◽

Nod Mice ◽

Treg Cells ◽

Immunological Tolerance ◽

Protein Vaccine ◽

Green Fluorescent

In animals, oral administration of the cholera toxin B (CTB) subunit conjugated to the autoantigen insulin enhances the specific immune-unresponsive state. This is called oral tolerance and is capable of suppressing autoimmune type 1 diabetes (T1D). However, the process by which the CTB-insulin (CTB-INS) protein works as a therapy for T1Din vivoremains unclear. Here, we successfully expressed a green fluorescent protein- (GFP-) tagged CTB-Ins (CTB-Ins-GFP) fusion protein in silkworms in a pentameric form that retained the native ability to activate the mechanism. Oral administration of the CTB-Ins-GFP protein induced special tolerance, delayed the development of diabetic symptoms, and suppressed T1D onset in nonobese diabetic (NOD) mice. Moreover, it increased the numbers of CD4+CD25+Foxp3+T regulatory (Treg) cells in peripheral lymph tissues and affected the biological activity of spleen cells. This study demonstrated that the CTB-Ins-GFP protein produced in silkworms acted as an oral protein vaccine, inducing immunological tolerance involving CD4+CD25+Foxp3+Treg cells in treating T1D.

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New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

Applied and Environmental Microbiology ◽

10.1128/aem.03677-15 ◽

2016 ◽

Vol 82 (8) ◽

pp. 2240-2246 ◽

Cited By ~ 10

Author(s):

Alex I. Kanno ◽

Cibelly Goulart ◽

Henrique K. Rofatto ◽

Sergio C. Oliveira ◽

Luciana C. C. Leite ◽

...

Keyword(s):

Mycobacterium Bovis ◽

Fluorescent Protein ◽

Vaccine Development ◽

Expression Vectors ◽

Content Type ◽

Expression Levels ◽

Wide Range ◽

Mycobacteriophage L5 ◽

Green Fluorescent ◽

Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

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Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein

The Journal of Cell Biology ◽

10.1083/jcb.139.6.1465 ◽

1997 ◽

Vol 139 (6) ◽

pp. 1465-1476 ◽

Cited By ~ 167

Author(s):

Norio Sakai ◽

Keiko Sasaki ◽

Natsu Ikegaki ◽

Yasuhito Shirai ◽

Yoshitaka Ono ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Green Fluorescent Protein ◽

Nmda Receptor ◽

Fusion Protein ◽

Fluorescent Protein ◽

Living Cells ◽

Kinase C ◽

Gfp Fusion Protein ◽

Green Fluorescent

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.

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846 Pre-clinical validation of a FLT3L-fusion protein for dendritic cell expansion and anti-tumor efficacy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.846 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A887-A887

Author(s):

Michelle Kuhne ◽

Hamlet Chu ◽

Sarah Ng ◽

Christopher Clarke ◽

Brian Carr ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Fusion Protein ◽

Tyrosine Kinase ◽

Single Agent ◽

Tumor Growth Inhibition ◽

Cell Responses ◽

Dose Dependent ◽

Cell Death Protein

BackgroundThe ligand for the receptor tyrosine kinase FMS-like tyrosine kinase 3 (FLT3) plays an importantrole in hematopoiesis. FLT3 signaling is required for the differentiation andexpansion of dendritic cells. In the context of cancer immunity, the conventional dendritic cellsubtype 1 (cDC1) are required for the generation of tumor-specific T cell responses in mousepreclinical models. In human tumors cDC1 are often underrepresented in thetumor microenvironment, supporting the hypothesis that therapeutically increasing their number via FLT3 pathway stimulation has the potential to promote T cell-mediated anti-tumor efficacy.MethodsGS-3583 is a fusion protein composed of the extracellular domain (ECD) of human FLT3 ligand(FLT3L) combined with a modified fragment crystallizable (Fc) region of human IgG4. GS-3583was designed to induce cDC1 expansion and subsequently promote tumor-reactive T cell priming, activation and recruitment into the tumor microenvironment. To evaluate the therapeutic efficacy of FLT3 stimulation in vivo, a mouse surrogate mGS-3583was designed using the ECD of mouse FLT3L fused to an engineered mouse IgG2a Fc withattenuated binding to mouse FcgRs.Results mGS-3583 bound to recombinant mouse FLT3 with an estimated affinity of 15 nM, and to mouse FLT3-expressing cells with an EC50 of 0.15 nM. In vivo, mGS-3583 showed single agent dose-dependent tumor growth inhibition (TGI) in tumors that correlated with peripheral and intratumoral cDC1 expansion. In tumors with no initial immune infiltration, mGS-3583 led to an influx of T cells into the tumors. In addition to single agent efficacy, mGS-3583 combined effectively with programmed cell death protein (ligand)-1 (PD(L)-1) pathway blockade.ConclusionsIn vivo expansion of dendritic cells can convert uninflamed (cold) tumors to immunologically active (hot) tumors and initiate productive anti-tumor immune responses. These findings support the development GS-3583 as a promising candidate for cancer immunotherapy.

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