Poxvirus vaccine strategies to improve T cell responses: Neutrophil immunomodulation & promoter modification

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-05-2015Vaccinia virus (VACV) is being used as vaccine vector for several animal and human diseases. Its capacity to induce specific T cell responses to foreign antigens define the best immune property of this virus, which generates protection against distinct pathogens. The MVA and NYVAC virus strains, which have limited replication in human cells, are the most-used VACV vector in clinical trials. This study is directed to understanding the poxvirus-dependent mechanisms involved in the generation of antigen-specific immune responses, and to improving poxvirus capacity to induce this response. Here we developed different strategies and distinct VACV-based vaccine candidates to improve foreign antigen-specific immune responses. We modified NYVAC-C that expressed HIV-1 clade C antigens by gene deletion (A52R, K7R, B15R) to target the NFκB central host-cell signaling pathway. We found that these genes act together to inhibit NFκB signaling, ruling out synergy between these proteins as a mode of action. Our study of the ability of this recombinant virus to induce NFκB-dependent innate immune responses demonstrated that neutrophils increase the T cell adaptive immune response to HIV antigens. We tracked the neutrophil trafficking from the infection site to various secondary lymphoid organs and showed that, after chemokine stimulation, neutrophils polarize to distinct functional phenotypes that regulate the magnitude and quality of the immune response. We generated new VACV promoters to improve the timing of GFP (green fluorescent protein) and LACK (Leishmania homologue of receptors for activated Ckinase) expression and to increase antigen-specific T cell responses. Using bioinformatics analysis, we defined a new early promoter motif to improve GFP antigen early expression. Furthermore, we modified the promoter spacer length to increase this heterologous antigen expression. The recombinant MVA vectors with these promoter modifications increased heterologous gene expression and positively influenced the magnitude and the quality of antigen-specific T cell memory responses. These findings provide important insights into the mechanism of the VACV-induced immune response and alternative strategies for vaccine vector design

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy

Validation of Two Commercial Multiplex Real-Time PCR Assays for Detection of SARS-CoV-2 in Stool Donors for Fecal Microbiota Transplantation

Recurrent infection by Clostridioides difficile has recently been treated by fecal microbiota transplantation (FMT). As viable SARS-CoV-2 was recovered from stool of asymptomatic individuals, the FMT procedure could be a potential risk of SARS-CoV-2 transmission, thus underlying the need to reliably detect SARS-CoV-2 in stool. Here, we performed a multicentric study to explore performances of two commercially available assays for detection of SARS-CoV-2 RNA in stool of potential FMT donors. In three hospitals, 180 stool samples were spiked with serial 10-fold dilutions of a SARS-CoV-2 inactivated lysate to evaluate the Seegene Allplex ™ SARS-CoV-2 (SC2) and SARS-CoV- 2/FluA/FluB/RSV (SC2FABR) Assays for the detection of viral RNA in stool of FMT donors. The results revealed that both assays detected down to 2 TCID50/mL with comparable limit of detection values, SC2 showing more consistent target positivity rate than SC2FABR. Beyond high amplification efficiency, correlation between CT values and log concentrations of inactivated viral lysates showed R2 values ranging from 0.88 to 0.90 and from 0.87 to 0.91 for the SC2 and SC2FABR assay, respectively. The present results demonstrate that both methods are highly reproducible, sensitive, and accurate for SARS-CoV-2 RNA detection in stool, suggesting a potential use in FMT-donor screening

Distinct p21 requirements for regulating normal and self-reactive T cells through IFN-γ production.

Self/non-self discrimination characterizes immunity and allows responses against pathogens but not self-antigens. Understanding the principles that govern this process is essential for designing autoimmunity treatments. p21 is thought to attenuate autoreactivity by limiting T cell expansion. Here, we provide direct evidence for a p21 role in controlling autoimmune T cell autoreactivity without affecting normal T cellresponses. We studied C57BL/6, C57BL/6/lpr and MRL/lpr mice overexpressing p21 in T cells, and showed reduced autoreactivity and lymphadenopathy in C57BL/6/lpr, and reduced mortality in MRL/lpr mice. p21 inhibited effector/memory CD4(+) CD8(+) and CD4(-)CD8(-) lpr T cell accumulation without altering defective lpr apoptosis. This was mediated by a previously non-described p21 function in limiting T cell overactivation and overproduction of IFN-γ, a key lupus cytokine. p21 did not affect normal T cell responses, revealing differential p21 requirements for autoreactive and normal T cell activity regulation. The underlying concept of these findings suggests potential treatments for lupus and autoimmune lymphoproliferative syndrome, without compromising normal immunity.This work was supported by grants from the Ministry of Economy and Competitivity (MINECO)/Instituto Carlos III (PI081835 PI11/00950) and the CAM (MITIC S2011/ BMD2502) to DB, and from the MINECO (SAF2010-21205 and PIB2010BZ-00564) and the CAM (MITIC S2011/BMD2502) to CMA.Peer reviewe

Translational Studies Using the MALT1 Inhibitor (S)-Mepazine to Induce Treg Fragility and Potentiate Immune Checkpoint Therapy in Cancer

INTRODUCTION: Regulatory T cells (Tregs) play a critical role in the maintenance of immune homeostasis but also protect tumors from immune-mediated growth control or rejection and pose a significant barrier to effective immunotherapy. Inhibition of MALT1 paracaspase activity can selectively reprogram immune-suppressive Tregs in the tumor microenvironment to adopt a proinflammatory fragile state, which offers an opportunity to impede tumor growth and enhance the efficacy of immune checkpoint therapy (ICT). METHODS: We performed preclinical studies with the orally available allosteric MALT1 inhibitor (S)-mepazine as a single-agent and in combination with anti-programmed cell death protein 1 (PD-1) ICT to investigate its pharmacokinetic properties and antitumor effects in several murine tumor models as well as patient-derived organotypic tumor spheroids (PDOTS). RESULTS: (S)-mepazine demonstrated significant antitumor effects and was synergistic with anti-PD-1 therapy in vivo and ex vivo but did not affect circulating Treg frequencies in healthy rats at effective doses. Pharmacokinetic profiling revealed favorable drug accumulation in tumors to concentrations that effectively blocked MALT1 activity, potentially explaining preferential effects on tumor-infiltrating over systemic Tregs. CONCLUSIONS: The MALT1 inhibitor (S)-mepazine showed single-agent anticancer activity and presents a promising opportunity for combination with PD-1 pathway-targeted ICT. Activity in syngeneic tumor models and human PDOTS was likely mediated by induction of tumor-associated Treg fragility. This translational study supports ongoing clinical investigations (ClinicalTrials.gov Identifier: NCT04859777) of MPT-0118, (S)-mepazine succinate, in patients with advanced or metastatic treatment-refractory solid tumors

DPY30 loss leads to DNA re-replication and immunoediting in pancreatic ductal adenocarcinoma

View full abstracthttps://openworks.mdanderson.org/leading-edge/1036/thumbnail.jp

Behavioral immune landscapes of inflammation.

Transcriptional or proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues. These approaches, however, fail to describe dynamic scenarios in which cells can change their biochemical properties and downstream “behavioral” outputs every few seconds or minutes. Here, we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamism of individual leukocytes at sites of active inflammation. By analyzing over 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioral descriptors of individual cells and used these high-dimensional datasets to build behavioral landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and inside blood vessels uncovered a continuum of neutrophil states, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioral in vivo screening of thousands of cells from 24 different mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and genetic or pharmacological interference of Fgr protected from inflammatory injury. Thus, behavioral landscapes report unique biological properties of dynamic environments at high cellular, spatial and temporal resolution.pre-print4302 K

The instrument control unit of the PLATO payload: design consolidation following the preliminary design review by ESA

PLATO is an M-class mission (M3) of the European Space Agency (ESA) whose launch is scheduled in 2026. The main aim of the mission is the detection and characterization of terrestrial exoplanets orbiting around bright solar-type star. The payload consists of 26 small telescopes: 24 "normal" cameras and 2 "fast" cameras. The huge amount of data produced by the PLATO telescopes is acquired and processed on-board by the Data Processing System (DPS) made up by various processing electronic units. The DPS of the PLATO instrument comprises the Normal and Fast DPUs (Data Processing Units) and a single ICU (Instrument Control Unit), are data routed through a SpaceWire network. The topic of this paper is the description of the architecture of the ICU and its role within the DPS, the status of the Avionic Validation Model (AVM) testing at the end of the Unit Preliminary Design Review (UPDR) performed by ESA and the results of the test of the first engineering model

Observation of the Production of Three Massive Gauge Bosons at root s=13 TeV

The first observation is reported of the combined production of three massive gauge bosons (VVV with V = W, Z) in proton-proton collisions at a center-of-mass energy of 13 TeV. The analysis is based on a data sample recorded by the CMS experiment at the CERN LHC corresponding to an integrated luminosity of 137 fb(-1). The searches for individualWWW, WWZ, WZZ, and ZZZ production are performed in final states with three, four, five, and six leptons (electrons or muons), or with two same-sign leptons plus one or two jets. The observed (expected) significance of the combinedVVV production signal is 5.7 (5.9) standard deviations and the corresponding measured cross section relative to the standard model prediction is 1.02(-0.23)(+0.26). The significances of the individual WWW and WWZ production are 3.3 and 3.4 standard deviations, respectively. Measured production cross sections for the individual triboson processes are also reported

Measurement of the W gamma Production Cross Section in Proton-Proton Collisions at root s=13 TeV and Constraints on Effective Field Theory Coefficients

A fiducial cross section for W gamma production in proton-proton collisions is measured at a center-of-mass energy of 13 TeV in 137 fb(-1) of data collected using the CMS detector at the LHC. The W -> e nu and mu nu decay modes are used in a maximum-likelihood fit to the lepton-photon invariant mass distribution to extract the combined cross section. The measured cross section is compared with theoretical expectations at next-to-leading order in quantum chromodynamics. In addition, 95% confidence level intervals are reported for anomalous triple-gauge couplings within the framework of effective field theory.Peer reviewe