scholarly journals Efficacy of Intracellular Activated Promoters for Generation of Salmonella-Based Vaccines

2010 ◽  
Vol 78 (11) ◽  
pp. 4828-4838 ◽  
Author(s):  
Xin Xu ◽  
Mohamed I. Husseiny ◽  
Andreas Goldwich ◽  
Michael Hensel
Keyword(s):  
T Cells ◽  
Rational Design ◽  
Optimal Level ◽  
Effector Proteins ◽  
Vaccine Antigen ◽  
Antigen Delivery ◽  
Secretion Systems ◽  
Heterologous Antigens ◽  
Stimulation Of

ABSTRACT Salmonella enterica is a versatile vaccine carrier for heterologous antigens. One strategy for vaccine antigen delivery is the use of live attenuated S. enterica strains that translocate heterologous antigens into antigen-presenting cells by means of type III secretion systems (T3SS). The feasibility of this approach has been demonstrated in various experimental vaccination studies. The efficacy of recombinant live vaccines is critically influenced by the optimal level of attenuation and many other factors. For the rational design of approaches involving translocation by T3SS, additional parameters are the level of expression of the heterologous antigens and the selection of carrier proteins for the delivery of antigens to desirable subcellular compartments of the target cell. We deployed the Salmonella pathogenicity island 2 (SPI2)-encoded T3SS for antigen delivery. The SPI2-T3SS and effector proteins are encoded by members of the large SsrAB regulon, including promoters with highly variable strength of expression. We investigated the effect of various in vivo-activated promoters of the SsrAB regulon on the efficacy of recombinant Salmonella vaccines. We observed that the use of promoters with higher strength results in greater synthesis of recombinant antigens and greater stimulation of T-cell responses in cell culture assays for the stimulation of T cells by the model antigen ovalbumin. In contrast, in vaccination experiments, promoters with a low level of expression resulted in the induction of higher amounts of T cells reactive to the model antigen listeriolysin. These results demonstrate that high-level expression of heterologous antigens does not necessarily result in optimal stimulation of immune responses.

scholarly journals Tandem Translation Generates a Chaperone for the Salmonella Type III Secretion System Protein SsaQ

2011 ◽  
Vol 286 (41) ◽  
pp. 36098-36107 ◽  
Author(s):  
Xiu-Jun Yu ◽  
Mei Liu ◽  
Steve Matthews ◽  
David W. Holden
Keyword(s):  
Type Iii Secretion ◽  
Ex Vivo ◽  
Eukaryotic Cell ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Function Loss ◽  
Bacterial Cytoplasm

Type III secretion systems (T3SSs) of bacterial pathogens involve the assembly of a surface-localized needle complex, through which translocon proteins are secreted to form a pore in the eukaryotic cell membrane. This enables the transfer of effector proteins from the bacterial cytoplasm to the host cell. A structure known as the C-ring is thought to have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of the T3SS. Here, we studied SsaQ, an FliN-like putative C-ring protein of the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS. ssaQ produces two proteins by tandem translation: a long form (SsaQL) composed of 322 amino acids and a shorter protein (SsaQS) comprising the C-terminal 106 residues of SsaQL. SsaQL is essential for SPI-2 T3SS function. Loss of SsaQS impairs the function of the T3SS both ex vivo and in vivo. SsaQS binds to its corresponding region within SsaQL and stabilizes the larger protein. Therefore, SsaQL function is optimized by a novel chaperone-like protein, produced by tandem translation from its own mRNA species.

Existence and immunological significance of circulating Ia+T cells in autoimmune thyroid diseases

1987 ◽  
Vol 115 (2) ◽  
pp. 282-288 ◽  
Author(s):  
Kazuya Zeki ◽  
Takashi Fujihira ◽  
Fumihiko Shirakawa ◽  
Kenichi Watanabe ◽  
Sumiya Eto
Keyword(s):  
T Cells ◽  
Thyroid Diseases ◽  
Cytotoxicity Test ◽  
Autoimmune Thyroid Diseases ◽  
Autoimmune Thyroid ◽  
Hla Dr ◽  
Specific Antigens ◽  
Cellular Immune ◽  
Stimulation Of

Abstract. We investigated the percentage of circulating HLA-DR antigen positive (Ia antigen positive: Ia+) T cells and the additive proliferation by non-specific mitogens and thyroid-specific antigens by means of a cytotoxicity test in autoimmune thyroid diseases. Furthermore, we studied the stimulative function of circulating Ia+T cells in autologous mixed lymphocyte reactions. %Ia+T cells were significantly increased in patients with autoimmune thyroid diseases compared with those in normal controls. They were additionally increased by the stimulation of TSH-receptor or thyroid-microsome in patients with Graves' disease, and by the stimulation of thyroglobulin and thyroid-microsome in patients with Hashimoto's thyroiditis. As to the cellular immune function, circulating Ia+T cells stimulated Ia− T cells in autologous MLR in patients with autoimmune thyroid diseases. These data suggest that some of the T cells are already activated in vivo, that the activation of T cells may be by thyroid-specific antigens, and that these activated (Ia+) T cells may be able sequentially to induce the activation of inactivated (Ia−) T cells in autoimmune thyroid diseases.

Stimulation of B and T cells by in vivo high dose immunoglobulin administration in normal mice

1991 ◽  
Vol 4 (2) ◽  
pp. 325-339 ◽  
Author(s):  
Anne Sundblad ◽  
François Huetz ◽  
Denis Portnoï ◽  
Antonio Coutinho
Keyword(s):  
T Cells ◽  
High Dose ◽  
Stimulation Of

Leukemia-Reactive Virus-Specific T Cells Generated by T Cell Receptor (TCR) Transfer Preserve Their Dual Specificity upon Repetitive Stimulation of the Endogenous TCR.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 136-136
Author(s):  
M.M. van Loenen ◽  
R.S. Hagedoorn ◽  
M. Hoogeboom ◽  
M.G.D. Kester ◽  
Roelof Willemze ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Repetitive Stimulation ◽  
Cell Surface Expression ◽  
Viral Antigens ◽  
Low Expression ◽  
Stimulation Of

Abstract TCR-transfer to engineer tumor-specific T cells may be a strategy for adoptive immunotherapy. For complete eradication of leukemic cells and to achieve long-term protection, potent effector T cell function and long-term T cell persistence are necessary. Therefore, we propose to use virus specific T cells for TCR transfer since such engineered dual specific T cells can be triggered via their endogenous TCR by latent presence of viral antigens, improving their long-term persistence. We have previously shown that virus specific T cells can be redirected towards anti-leukemic reactivity by transfer of the hematopoietic minor histocompatibility antigen HA-2 specific TCR (HA-2-TCR). The TCR-transferred virus specific T cells showed differences in TCR cell surface make up, which was stable for months after repetitive non-specific TCR triggering. The T cells expressed either both TCRs intermediately at the cell surface, or the endogenous TCR was highly expressed with a low expression of the introduced TCR, or the introduced TCR was highly expressed with a low expression of the endogenous TCR. It may be anticipated that frequent encounter with viral antigens in vivo leads to selective outgrowth of TCR-transferred dual specific T cells with high expression of the endogenous viral specific TCR but low expression of the introduced tumor specific TCR, resulting in reduced anti-leukemic reactivity. To address this issue, we generated CMVA2-specific T cells transduced with the HA-2-TCR. This resulted in dual specific cells with different TCR cell surface make up. The dual specific T cells were repetitively stimulated specifically either via their endogenous virus specific TCR or via the introduced HA-2 specific TCR. In time, the cell surface expression of the endogenous and introduced TCRs as measured with CMVA2 and HA-2A2 tetramers diverged. Repetitive stimulation of the endogenous TCR skewed the dual specific T cells towards a cell population that predominantly expressed the endogenous TCR. In contrast, repetitive stimulation of the introduced TCR skewed the cells towards T cells that predominantly expressed the introduced TCR. However, this divergence in tetramer stainings was shown to quickly revert after a single stimulation via the other TCR. To study whether this divergence was the result of a difference in TCR cell surface distribution or of selective outgrowth of different T cells, T cells were sorted that predominantly expressed either the endogenous or the introduced TCR. These cells were subsequently stimulated on the endogenous or introduced TCR, and compared regarding TCR cell surface expression and functional activity. Directly after sorting dual specific T cells preferentially expressing the endogenous TCR were still reactive against HA-2+ target cells, although the reactivity was reduced compared to cells preferentially expressing the introduced TCR. However, when restimulated on the introduced HA-2-TCR, the dual specific T cells expanded antigen specifically, and reverted within several days into cells with high expression of the introduced TCR that exerted potent HA-2 specific anti-leukemic effector functions. In conclusion, we demonstrate that these dual specific T cells are likely to persist in vivo due to repetitive encounter with viral antigens with preservation of anti-leukemic effector function. Moreover, in vivo exposure to the tumor associated antigen will further enhance the relevant specificity.

scholarly journals Type I Interferon-mediated Stimulation of T Cells by CpG DNA

1998 ◽  
Vol 188 (12) ◽  
pp. 2335-2342 ◽  
Author(s):  
Siquan Sun ◽  
Xiaohong Zhang ◽  
David F. Tough ◽  
Jonathan Sprent
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Type I Interferons ◽  
Type I ◽  
Cpg Dna ◽  
Stimulation Of

Immunostimulatory DNA and oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA) are strongly stimulatory for B cells and antigen-presenting cells (APCs). We report here that, as manifested by CD69 and B7-2 upregulation, CpG DNA also induces partial activation of T cells, including naive-phenotype T cells, both in vivo and in vitro. Under in vitro conditions, CpG DNA caused activation of T cells in spleen cell suspensions but failed to stimulate highly purified T cells unless these cells were supplemented with APCs. Three lines of evidence suggested that APC-dependent stimulation of T cells by CpG DNA was mediated by type I interferons (IFN-I). First, T cell activation by CpG DNA was undetectable in IFN-IR−/− mice. Second, in contrast to normal T cells, the failure of purified IFN-IR−/− T cells to respond to CpG DNA could not be overcome by adding normal IFN-IR+ APCs. Third, IFN-I (but not IFN-γ) caused the same pattern of partial T cell activation as CpG DNA. Significantly, T cell activation by IFN-I was APC independent. Thus, CpG DNA appeared to stimulate T cells by inducing APCs to synthesize IFN-I, which then acted directly on T cells via IFN-IR. Functional studies suggested that activation of T cells by IFN-I was inhibitory. Thus, exposing normal (but not IFN-IR−/−) T cells to CpG DNA in vivo led to reduced T proliferative responses after TCR ligation in vitro.

scholarly journals Dormant Intracellular Salmonella enterica Serovar Typhimurium Discriminates among Salmonella Pathogenicity Island 2 Effectors To Persist inside Fibroblasts

2013 ◽  
Vol 82 (1) ◽  
pp. 221-232 ◽  
Author(s):  
Cristina Núñez-Hernández ◽  
Ana Alonso ◽  
M. Graciela Pucciarelli ◽  
Josep Casadesús ◽  
Francisco García-del Portillo
Keyword(s):  
Salmonella Enterica ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Intracellular Bacteria ◽  
Secretion Systems ◽  
Cultured Fibroblasts ◽  
Content Type ◽  
Type Iii Secretion Systems ◽  
Serovar Typhimurium

ABSTRACTSalmonella entericauses effector proteins delivered by type III secretion systems (TTSS) to colonize eukaryotic cells. Recentin vivostudies have shown that intracellular bacteria activate the TTSS encoded bySalmonellapathogenicity island-2 (SPI-2) to restrain growth inside phagocytes. Growth attenuation is also observedin vivoin bacteria colonizing nonphagocytic stromal cells of the intestinal lamina propria and in cultured fibroblasts. SPI-2 is required for survival of nongrowing bacteria persisting inside fibroblasts, but its induction mode and the effectors involved remain unknown. Here, we show that nongrowing dormant intracellular bacteria use the two-component system OmpR-EnvZ to induce SPI-2 expression and the PhoP-PhoQ system to regulate the time at which induction takes place, 2 h postentry. Dormant bacteria were shown to discriminate the usage of SPI-2 effectors. Among the effectors tested, SseF, SseG, and SseJ were required for survival, while others, such as SifA and SifB, were not. SifA and SifB dispensability correlated with the inability of intracellular bacteria to secrete these effectors even when overexpressed. Conversely, SseJ overproduction resulted in augmented secretion and exacerbated bacterial growth. Dormant bacteria produced other effectors, such as PipB and PipB2, that, unlike what was reported for epithelial cells, did not to traffic outside the phagosomal compartment. Therefore, permissiveness for secreting only a subset of SPI-2 effectors may be instrumental for dormancy. We propose that theS. entericaserovar Typhimurium nonproliferative intracellular lifestyle is sustained by selection of SPI-2 effectors that are produced in tightly defined amounts and delivered to phagosome-confined locations.

scholarly journals Dendritic Cell-Specific Antigen Delivery by Coronavirus Vaccine Vectors Induces Long-Lasting Protective Antiviral and Antitumor Immunity

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Luisa Cervantes-Barragan ◽  
Roland Züst ◽  
Reinhard Maier ◽  
Sophie Sierro ◽  
Jozef Janda ◽  
...  
Keyword(s):  
T Cells ◽  
Specific Antigen ◽  
Antigen Delivery ◽  
Vaccine Vectors ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Human Coronavirus 229E

ABSTRACTEfficient vaccination against infectious agents and tumors depends on specific antigen targeting to dendritic cells (DCs). We report here that biosafe coronavirus-based vaccine vectors facilitate delivery of multiple antigens and immunostimulatory cytokines to professional antigen-presenting cellsin vitroandin vivo. Vaccine vectors based on heavily attenuated murine coronavirus genomes were generated to express epitopes from the lymphocytic choriomeningitis virus glycoprotein, or human Melan-A, in combination with the immunostimulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). These vectors selectively targeted DCsin vitroandin vivoresulting in vector-mediated antigen expression and efficient maturation of DCs. Single application of only low vector doses elicited strong and long-lasting cytotoxic T-cell responses, providing protective antiviral and antitumor immunity. Furthermore, human DCs transduced with Melan-A-recombinant human coronavirus 229E efficiently activated tumor-specific CD8+T cells. Taken together, this novel vaccine platform is well suited to deliver antigens and immunostimulatory cytokines to DCs and to initiate and maintain protective immunity.IMPORTANCEVaccination against infectious agents has protected many individuals from severe disease. In addition, prophylactic and, most likely, also therapeutic vaccination against tumors will save millions from metastatic disease. This study describes a novel vaccine approach that facilitates delivery of viral or tumor antigens to dendritic cellsin vivo. Concomitant immunostimulation via the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) was achieved through delivery by the same viral vector. Single immunization with only low doses of coronavirus-based vaccine vectors was sufficient to elicit (i) vigorous expansion and optimal differentiation of CD8+T cells, (ii) protective and long-lasting antiviral immunity, and (iii) prophylactic and therapeutic tumor immunity. Moreover, highly efficient antigen delivery to human DCs with recombinant human coronavirus 229E and specific stimulation of human CD8+T cells revealed that this approach is exceptionally well suited for translation into human vaccine studies.

scholarly journals A new family of Type VI secretion system-delivered effector proteins displays ion-selective pore-forming activity

2019 ◽  
Author(s):  
Giuseppina Mariano ◽  
Katharina Trunk ◽  
David J. Williams ◽  
Laura Monlezun ◽  
Henrik Strahl ◽  
...  
Keyword(s):  
Effector Proteins ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
New Family ◽  
Type Vi Secretion ◽  
Outer Membrane Permeability ◽  
Polymicrobial Communities ◽  
Bacterial Effectors

AbstractType VI secretion systems (T6SSs) are nanomachines widely used by bacteria to compete with rivals. T6SSs deliver multiple toxic effector proteins directly into neighbouring cells and play key roles in shaping diverse polymicrobial communities. A number of families of T6SS-dependent anti-bacterial effectors have been characterised, however the mode of action of others remains unknown. Here we report that Ssp6, an anti-bacterial effector delivered by theSerratia marcescensT6SS, is an ion-selective pore-forming toxin.In vivo, Ssp6 inhibits growth by causing depolarisation of the inner membrane of intoxicated cells and also leads to increased outer membrane permeability, whilst reconstruction of Ssp6 activityin vitrodemonstrated that it forms cation-selective pores. A survey of bacterial genomes revealed that Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 represents a new family of T6SS-delivered anti-bacterial effectors, further diversifying the portfolio of weapons available for deployment during inter-bacterial conflict.

scholarly journals Transcriptional profiling reveals T cells cluster around neurons injected with Toxoplasma gondii proteins

2020 ◽  
Author(s):  
Emily F. Merritt ◽  
Hannah J. Johnson ◽  
Z. Sheen Wong ◽  
Adam S. Buntzman ◽  
Austin C. Conklin ◽  
...  
Keyword(s):  
T Cells ◽  
Toxoplasma Gondii ◽  
Transcriptional Profiling ◽  
Effector Proteins ◽  
Symptomatic Disease ◽  
Host Cell Interactions ◽  
And Control ◽  
Laser Capture

AbstractToxoplasma gondii’s tropism for and persistence in the CNS underlies the symptomatic disease Toxoplasma causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and infects in vivo. This predilection for neurons suggests that Toxoplasma’s persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on Toxoplasma-host cell interactions has been done in vitro and in non-neuronal cells. We address this gap by utilizing our Toxoplasma-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA-seq, we isolated and transcriptionally profiled Toxoplasma-injected neurons (TINs), Bystander neurons (nearby non-Toxoplasma injected neurons), and neurons from uninfected mice (controls). These profiles show that TINs transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that Toxoplasma transcripts were primarily found in the TINs transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells.

scholarly journals Salmonella Pathogenicity Island 2-Mediated Overexpression of Chimeric SspH2 Proteins for Simultaneous Induction of Antigen-Specific CD4 and CD8 T Cells

2005 ◽  
Vol 73 (1) ◽  
pp. 334-341 ◽  
Author(s):  
Klaus Panthel ◽  
Katrin M. Meinel ◽  
Victòria E. Sevil Domènech ◽  
Heike Retzbach ◽  
Emeka I. Igwe ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Protein Delivery ◽  
Effector Proteins ◽  
Foreign Protein ◽  
Secretion Systems ◽  
Heterologous Antigen ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Carrier Molecule

ABSTRACT Salmonella enterica serovar Typhimurium employs two different type III secretion systems (TTSS) encoded within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) for targeting of effector proteins into the cytosol of eukaryotic cells during different stages of the infection cycle. The SPI1 TTSS translocates virulence factors across the plasma membrane when the bacterium initially contacts the host cell. In contrast, the SPI2 TTSS functions to translocate proteins across the membrane of the Salmonella-containing vacuole and promotes intracellular survival and replication. The aim of the present study was to directly compare the potentials of SPI1 and SPI2 type III effector proteins to act as carrier molecules for a heterologous antigen. The p60 protein of Listeria monocytogenes was used as a model antigen to construct chimeric SopE2 (SPI1), SifA (SPI2), and SspH2 (SPI2) proteins. SPI1- and SPI2-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of p60 fusion proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with recombinant Salmonella strains expressing these hybrid proteins revealed comparable numbers of p60-specific CD8 T cells. However, only overexpression of translocated SspH2/p60 from a medium-copy-number vector induced simultaneous antigen-specific CD4 and CD8 T-cell responses, suggesting that SspH2 is an attractive carrier molecule for foreign-protein delivery.

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