Modulation of Cell Surface Receptor Expression by Modified Vaccinia Virus Ankara in Leukocytes of Healthy and HIV-Infected Individuals

Viral vectors are increasingly used as delivery means to induce a specific immunity in humans and animals. However, they also impact the immune system, and it depends on the given context whether this is beneficial or not. The attenuated vaccinia virus strain modified vaccinia virus Ankara (MVA) has been used as a viral vector in clinical studies intended to treat and prevent cancer and infectious diseases. The adjuvant property of MVA is thought to be due to its capability to stimulate innate immunity. Here, we confirmed that MVA induces interleukin-8 (IL-8), and this chemokine was upregulated significantly more in monocytes and HLA-DR(bright)dendritic cells (DCs) of HIV-infected patients on combined antiretroviral therapy (ART) than in cells of healthy persons. The effect of MVA on cell surface receptors is mostly unknown. Using mass cytometry profiling, we investigated the expression of 17 cell surface receptors in leukocytes afterex vivoinfection of human whole-blood samples with MVA. We found that MVA downregulates most of the characteristic cell surface markers in particular types of leukocytes. In contrast, C-X-C motif chemokine receptor 4 (CXCR4) was significantly upregulated in each leukocyte type of healthy persons. Additionally, we detected a relative higher cell surface expression of the HIV-1 co-receptors C-C motif chemokine receptor 5 (CCR5) and CXCR4 in leukocytes of HIV-ART patients than in healthy persons. Importantly, we showed that MVA infection significantly downregulated CCR5 in CD4+ T cells, CD8+ T cells, B cells, and three different DC populations. CD86, a costimulatory molecule for T cells, was significantly upregulated in HLA-(DRDCs)-D-bright after MVA infection of whole blood from HIV-ART patients. However, MVA was unable to downregulate cell surface expression of CD11b and CD32 in monocytes and neutrophils of HIV-ART patients to the same extent as in monocytes and neutrophils of healthy persons. In summary, MVA modulates the expression of many different kinds of cell surface receptors in leukocytes, which can vary in cells originating from persons previously infected with other pathogens

Prospective Observational Study on acute Appendicitis Worldwide (POSAW)

Acute appendicitis (AA) is the most common surgical disease, and appendectomy is the treatment of choice in the majority of cases. A correct diagnosis is key for decreasing the negative appendectomy rate. The management can become difficult in case of complicated appendicitis. The aim of this study is to describe the worldwide clinical and diagnostic work-up and management of AA in surgical departments.info:eu-repo/semantics/publishedVersio

Immunological markers to predict vaccine safety

La vaccination est souvent mal perçue par la population générale. Pour rassurer cette dernière, il serait intéressant de créer une plateforme vaccinale pouvant prédire, in vitro, les risques associés à la prise d’un vaccin. L’objectif de cette thèse est de mettre au point les prémices de cette plateforme. Le principe est simple: obtenir la signature inflammatoire d’un vaccin candidat pour évaluer son innocuité. Pour cela, cette signature sera comparée à celles obtenues par des vaccins actuellement sur le marché ou induites par des pathogènes.Durant cette thèse, nous avons sélectionné une liste de biomarqueurs pouvant être utilisés pour déterminer la signature inflammatoire d’un vaccin. Pour mettre au point cette liste, nous avons utilisé différents modèles inflammatoires (VIH et ligands TLR) et la cytométrie de masse. Par la suite, nous avons mis au point des tests in vitro pour obtenir les signatures inflammatoires induites par le Vaccinia Virus ou le Modified Vaccinia virus Ankara, tous deux utilisés pour éradiquer la variole. Nous avons identifié des signatures inflammatoires spécifiques pour chacun de ces virus, à la fois chez des individus sains et chez des patients infectés par le VIH.La poursuite de ces études, par l’obtention d’un grand nombre de signatures provenant de vaccins sur le marché ou induites par des pathogènes, pourrait permettre de finaliser la mise en place de cette plateforme. En effet, l’obtention de ces dernières permettrait d’obtenir des signatures de référence qui pourraient prédire la dangerosité d’un vaccin.Vaccination is often not well regarded by the general population. To reassure this latest, it will be interesting to set up an in vitro platform predicting the vaccine safety. The aim of this thesis is to develop the beginnings of this platform. The principle is simple, to get inflammatory signature of a candidate vaccine to evaluate it safety. For that, this signature will be compared with those obtained by vaccine currently on the market or by pathogens.During this thesis, we selected a list of biomarkers that can be used to determinate the inflammatory signature of a vaccine. To obtain this list, we used different inflammatory models (HIV and TLR ligands) and the mass cytometry. Then, we had developed in vitro test to obtain inflammatory signatures induced by Vaccinia Virus or Modified Vaccinia virus Ankara, each used to eradicate the smallpox. We identified specific inflammatory signatures for each virus, both in healthy individuals and HIV-infected humans.The continuation of these studies, by obtaining a large number of signatures coming from vaccines on the market or induced by pathogens, could make it possible to finalize the setting up of this platform. Indeed, the obtaining of the latter would make it possible to obtain reference signatures which could predict the dangerousness of a vaccine

Characterization of Leukocytes From HIV-ART Patients Using Combined Cytometric Profiles of 72 Cell Markers.

International audienceMotivation: Mass cytometry is a technique used to measure the intensity levels of proteins expressed by cells, at a single cell resolution. This technique is essential to characterize the phenotypes and functions of immune cell populations, but is currently limited to the measurement of 40 cell markers that restricts the characterization of complex diseases. However, algorithms and multi-tube cytometry techniques have been designed for combining phenotypic information obtained from different cytometric panels. The characterization of chronic HIV infection represents a good study case for multi-tube mass cytometry as this disease triggers a complex interactions network of more than 70 cell markers. Method: We collected whole blood from non-viremic HIV-infected patients on combined antiretroviral therapies and healthy donors. Leukocytes from each individual were stained using three different mass cytometry panels, which consisted of 35, 32, and 33 cell markers. For each patient and using the CytoBackBone algorithm, we combined phenotypic information from three different antibody panels into a single cytometric profile, reaching a phenotypic resolution of 72 markers. These high-resolution cytometric profiles were analyzed using SPADE and viSNE algorithms to decipher the immune response to HIV. Results: We detected an upregulation of several proteins in HIV-infected patients relative to healthy donors using our profiling of 72 cell markers. Among them, CD11a and CD11b were upregulated in PMNs, monocytes, mDCs, NK cells, and T cells. CD11b was also upregulated on pDCs. Other upregulated proteins included: CD38 on PMNs, monocytes, NK cells, basophils, B cells, and T cells; CD83 on monocytes, mDCs, B cells, and T cells; and TLR2, CD32, and CD64 on PMNs and monocytes. These results were validated using a mass cytometry panel of 25 cells markers. Impacts: We demonstrate here that multi-tube cytometry can be applied to mass cytometry for exploring, at an unprecedented level of details, cell populations impacted by complex diseases. We showed that the monocyte and PMN populations were strongly affected by the HIV infection, as CD11a, CD11b, CD32, CD38, CD64, CD83, CD86, and TLR2 were upregulated in these populations. Overall, these results demonstrate that HIV induced a specific environment that similarly affected multiple immune cells

A high-resolution mass cytometry analysis reveals a delay of cytokines production after TLR4 or TLR7/8 engagements in HIV-1 infected humans

International audienceHIV infection is associated with chronic inflammation in both non-treated and treated patients. TLR-dependent mechanisms are strongly involved in the maintenance of this inflammation. Indeed, the residual replication of HIV, the potential viral co-infections, or the products issued from microbial translocation provide TLR ligands, which contribute to trigger innate immune responses. Maintaining this chronic inflammation leads to an exhaustion of the immune system. Therefore, the TLR-dependent responses could be altered in HIV-infected patients. To investigate this hypothesis, we performed high-resolution phenotyping using a mass cytometry panel of 34 cell markers. Whole blood cells from healthy, non-treated HIV-infected and ART-treated HIV-infected subjects were stimulated with LPS, R848 or Poly(I:C). We observed the immune responses induced in T-cells, Bcells, polymorphonuclear cells, NK cells, basophils, monocytes and dendritic cells. We observed that, for either LPS or R848 stimulations, the production of cytokines in monocytes and conventional dendritic cells was delayed in treated or non-treated HIV-infected patients, compared to healthy individuals. These results suggest that leukocytes from chronic HIV-infected patients are slower to respond following the sensing of pathogens and danger signals, which may be an important feature of HIV infection

Modulation of Cell Surface Receptor Expression by Modified Vaccinia Virus Ankara in Leukocytes of Healthy and HIV-Infected Individuals

Vaccines represent one of the major advances of modern medicine. Despite the many successes of vaccination, continuous efforts to design new vaccines are needed to fight “old” pandemics, such as tuberculosis and malaria, as well as emerging pathogens, such as Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccination aims at reaching sterilizing immunity, however assessing vaccine efficacy is still challenging and underscores the need for a better understanding of immune protective responses. Identifying reliable predictive markers of immunogenicity can help to select and develop promising vaccine candidates during early preclinical studies and can lead to improved, personalized, vaccination strategies. A systems biology approach is increasingly being adopted to address these major challenges using multiple high-dimensional technologies combined with in silico models. Although the goal is to develop predictive models of vaccine efficacy in humans, applying this approach to animal models empowers basic and translational vaccine research. In this review, we provide an overview of vaccine immune signatures in preclinical models, as well as in target human populations. We also discuss high-throughput technologies used to probe vaccine-induced responses, along with data analysis and computational methodologies applied to the predictive modeling of vaccine efficacy

Characterization of Phenotypes and Functional Activities of Leukocytes From Rheumatoid Arthritis Patients by Mass Cytometry.

International audienceBackground: Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease and leads to persistent chronic inflammation. The pathophysiology of the disease is complex, involving both adaptive and innate immunity. Among innate immune cells, neutrophils have been rarely studied due to their sensitivity to freezing and they are not being collected after Ficoll purification.Methods: We used mass cytometry to perform a multidimensional phenotypic characterization of immune cells from RA-treated patients, which included the simultaneous study of 33 intra- or extra-cellular markers expressed by leukocytes. We were able to focus our study on innate immune cells, especially neutrophils, due to a specific fixation method before freezing. In addition, blood samples were stimulated or not with various TLR agonists to evaluate whether RA-dependent chronic inflammation can lead to immune-cell exhaustion.Results: We show that RA induces the presence of CD11blow neutrophils (33.7 and 9.2% of neutrophils in RA and controls, respectively) associated with the duration of disease. This subpopulation additionally exhibited heterogeneous expression of CD16. We also characterized a CD11ahigh Granzyme Bhigh T-cell subpopulation possibly associated with disease activity. There was no difference in cytokine expression after the stimulation of immune cells by TLR agonists between RA and controls.Conclusion: Mass cytometry and our fixation method allowed us to identify two potential new blood subpopulations of neutrophils and T-cells, which could be involved in RA pathology. The phenotypes of these two potential new subpopulations need to be confirmed using other experimental approaches, and the exact role of these subpopulations is yet to be studied