Rational design of polymer-based nanosystems for an efficient targeting of the immune system

The main goal of this thesis is the development of polymer-based nanosystems for antigen delivery to the immune system. To evaluate the influence of the physicochemical characteristics of the nanosystems in their biodistribution to the lymphatic system, we have developed two chitosan nanocapsule prototypes with different particle size and the corresponding nanoemulsions, apart from other nanocapsules with different polymeric coatings. Results have shown the importance of particle size, surface charge and specific characteristics of the polymers used as coating in the draining of the nanocarriers to the lymphatic system. Moreover, we have developed chitosan:carboxymethyl-β-glucan nanoparticles, with high ability to associate the model antigen ovalbumin and an adequate storage stability profile, both in suspension and in dry powder form. Finally, in vivo studies have shown a significant accumulation of the formulation in the lymph nodes, leading to an efficient antigen presentation to T cells. Overall, our results show the potential of polymer-based systems to be modulated in terms of their composition, aiming at a more effective immune response

Nanoengineering of vaccines using natural polysaccharides

Currently, there are over 70 licensed vaccines, which prevent the pathogenesis of around 30 viruses and bacteria. Nevertheless, there are still important challenges in this area, which include the development of more active, non-invasive, and thermo-resistant vaccines. Important biotechnological advances have led to safer subunit antigens, such as proteins, peptides, and nucleic acids. However, their limited immunogenicity has demanded potent adjuvants that can strengthen the immune response. Particulate nanocarriers hold a high potential as adjuvants in vaccination. Due to their pathogen-like size and structure, they can enhance immune responses by mimicking the natural infection process. Additionally, they can be tailored for non-invasive mucosal administration (needle-free vaccination), and control the delivery of the associated antigens to a specific location and for prolonged times, opening room for single-dose vaccination. Moreover, they allow co-association of immunostimulatory molecules to improve the overall adjuvant capacity. The natural and ubiquitous character of polysaccharides, together with their intrinsic immunomodulating properties, their biocompatibility, and biodegradability, justify their interest in the engineering of nanovaccines. In this review, we aim to provide a state-of-the-art overview regarding the application of nanotechnology in vaccine delivery, with a focus on the most recent advances in the development and application of polysaccharide-based antigen nanocarriers

Mutation of the surface layer protein SlpB has pleiotropic effects in the probiotic propionibacterium freudenreichii CIRM-BIA 129

Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese-ripening starter, and currently considered as an emerging probiotic. As an example, the P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties. Its consumption accordingly exerts healing effects in different animal models of colitis, suggesting a potent role in the context of inflammatory bowel diseases. This anti-inflammatory effect depends on surface layer proteins (SLPs). SLPs may be involved in key functions in probiotics, such as persistence within the gut, adhesion to host cells and mucus, or immunomodulation. Several SLPs coexist in P. freudenreichii CIRM-BIA 129 and mediate immunomodulation and adhesion. A mutant P. freudenreichii CIRM-BIA 129ΔslpB (CB129ΔslpB) strain was shown to exhibit decreased adhesion to intestinal epithelial cells. In the present study, we thoroughly analyzed the impact of this mutation on cellular properties. Firstly, we investigated alterations of surface properties in CB129ΔslpB. Surface extractable proteins, surface charges (ζ-potential) and surface hydrophobicity were affected by the mutation. Whole-cell proteomics, using high definition mass spectrometry, identified 1,288 quantifiable proteins in the wild-type strain, i.e., 53% of the theoretical proteome predicted according to P. freudenreichii CIRM-BIA 129 genome sequence. In the mutant strain, we detected 1,252 proteins, including 1,227 proteins in common with the wild-type strain. Comparative quantitative analysis revealed 97 proteins with significant differences between wild-type and mutant strains. These proteins are involved in various cellular process like signaling, metabolism, and DNA repair and replication. Finally, in silico analysis predicted that slpB gene is not part of an operon, thus not affecting the downstream genes after gene knockout. This study, in accordance with the various roles attributed in the literature to SLPs, revealed a pleiotropic effect of a single slpB mutation, in the probiotic P. freudenreichii. This suggests that SlpB may be at a central node of cellular processes and confirms that both nature and amount of SLPs, which are highly variable within the P. freudenreichii species, determine the probiotic abilities of strains.Fil: do Carmo, Fillipe L. R.. Institut National de la Recherche Agronomique; Francia. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tavares, Guilherme C.. Universidade Federal de Minas Gerais; BrasilFil: Ibraim, Izabela C.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Cordeiro, Barbara F.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Oliveira, Emiliano R.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Rabah, Houem. Institut National de la Recherche Agronomique; FranciaFil: Cauty, Chantal. Institut National de la Recherche Agronomique; FranciaFil: da Silva, Sara H.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Canário Viana, Marcus V.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Caetano, Ana C. B.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: dos Santos, Roselane G.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: de Oliveira Carvalho, Rodrigo D.. Instituto de Ciencias Da Saúde; BrasilFil: Jardin, Julien. Institut National de la Recherche Agronomique; FranciaFil: Pereira, Felipe L.. Universidade Federal de Minas Gerais; BrasilFil: Folador, Edson L.. Universidade Estadual da Paraiba; BrasilFil: Le Loir, Yves. Institut National de la Recherche Agronomique; FranciaFil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; BrasilFil: Jan, Gwénaël. Institut National de la Recherche Agronomique; FranciaFil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Assembling immunomodulatory strains of Propionibacterium freudenreichii, Lactobacillus delbrueckii and Streptococcus thermophilus to produce an anti-inflammatory Emmental cheese

International audienceIntroduction and Aims. Inflammatory Bowel Diseases (IBD), including Ulcerative Colitis (UC), coincide with alterations in the gut microbiota. Consumption of immunomodulatory strains of probiotic bacteria may induce or prolong remission in UC patients. Fermented foods, including cheeses, constitute major vectors for bacteria consumption. New evidences revealed anti-inflammatory effects of selected strains within the species P.freudenreichii, S.thermophilus and L.delbrueckii. We thus hypothesized that consumption of a functional cheese, fermented by selected strains of these three species, may exert a positive effect on IBD.Methods. Strains of each bacterial species were screened based on the ability to induce regulatory IL-10 in human immune PBMC cells, or to downregulate nuclear factor kappa B (NF-κB) activation in a cultured HT-29 human intestinal epithelial cell line. We then investigated the effect of monostrain experimental cheese. We further investigated the impact two-strains experimental cheese. Finally, we produced in industrial conditions an Emmental cheese using one selected strain of each of the three species, i.e. P. freudenreichii CIRM-BIA 129 in combination with Lactobacillus delbrueckii CNRZ327 and Streptococcus thermophilus LMD-9. Consumption of all cheeses was investigated with respect to prevention of chemically induced colitis in mice.Results. Consumption of the experimental cheeses, or of the industrial Emmental, reduced the severity of subsequent chemically induced colitis, weight loss, disease activity index and histological score, in mice. Furtherrmore, consumption of the Emmental cheese, in a preventive way, reduced small bowel Immunoglobulin A (IgA) secretion, restored occludin gene expression and prevented induction of Tumor Necrosis Factor α (TNFα), Interferon γ (IFNγ) and Interleukin-17 (IL-17).Conclusion. Assembling immunomodulatory strains of both lactic acid and propionic acid starter bacteria leads to an anti-inflammatory Emmental cheese, as revealed in vivo. This opens new perspectives for the development of functional fermented food products for personalised nutrition in the context of IBD

Beneficial Propionibacteria within a Probiotic Emmental Cheese Impact on Dextran Sodium Sulphate-Induced Colitis in Mice

International audienceBackgrounds and aims - Inflammatory Bowel Diseases (IBD), including Ulcerative Colitis (UC), coincide with alterations in the gut microbiota. Consumption of immunomodulatory strains of probiotic bacteria may induce or prolong remission in UC patients. Fermented foods, including cheeses, constitute major vectors for bacteria consumption. New evidences revealed anti-inflammatory effects in selected strains of . We thus hypothesized that consumption of a functional cheese, fermented by such a strain, may exert a positive effect on IBD. Methods - We investigated the impact of cheese fermented by on gut inflammation. We developed an experimental single-strain cheese solely fermented by a selected immunomodulatory strain of , CIRM-BIA 129. We moreover produced, in industrial conditions, an Emmental cheese using the same strain, in combination with CNRZ327 and LMD-9, as starters. Consumption of both cheeses was investigated with respect to prevention of Dextran Sodium Sulphate (DSS)-induced colitis in mice. Results - Consumption of the single-strain experimental cheese, or of the industrial Emmental, both fermented by CIRM-BIA 129, reduced severity of subsequent DSS-induced colitis, weight loss, disease activity index and histological score. Both treatments, in a preventive way, reduced small bowel Immunoglobulin A (IgA) secretion, restored occludin gene expression and prevented induction of Tumor Necrosis Factor α (TNFα), Interferon γ (IFNγ) and Interleukin-17 (IL-17). Conclusions - A combination of immunomodulatory strains of starter bacteria can be used to manufacture an anti-inflammatory cheese, as revealed in an animal model of colitis. This opens new perspectives for personalised nutrition in the context of IBD

Fermented milks, using Lactobacillus casei or Propionibacterium freudenreichii, prevent mucositis, a side effect of chemotherapy, in mice

Mucositis, a common and often debilitating side effect of cancer chemotherapy, is a clinically important gastrointestinal inflammatory disease. It consists in a painful inflammation and ulceration of the mucosa lining the digestive tract, which may compromise proper nutrition of the patient, as well as termination of the treatment. It thus increases mortality and morbidity and contributes to rising health care costs. Its treatment is mainly supportive and often fails to relieve symptoms. Several studies indicate a role of the microbiota and suggest a healing effect of probiotic ingestion. Probiotic bacteria may maintain homeostasis and reduce side effects of chemotherapy toxicity. A major limit to probiotic bacteria anti-inflammatory effect is their susceptibility to digestive stresses and several studies indicate the key protective role of food matrices in increasing probiotics’ effect via the protection of bacteria against digestive constraints. In this study, we investigated the role of fermented milk, using Lactobacillus casei or Propionibacterium freudenreichii as probiotic starter, and of its fortification via addition of whey proteins.Matériels et Méthodes/Materials and methodsWe evaluated the role of whey protein isolate (WPI), when added to skim milk fermented by L. casei BL23 or by P. freudenreichii 138, as a protective matrix against in vitro stress challenges. In addition, we investigated in vivo the therapeutic effect of these fermented beverages in a murine model of mucositis induced by 5-Fluorouracil (5-FU). The monitored outcomes included weight loss, ileum histopathological score, villus height, crypt depth and number of mucus-producing goblet cells.Résultats principaux/Main ResultsThis study demonstrated that milk supplementation with 30% (w/v) of WPI increases the survival rate of both strains when challenged with acid, bile salts, high temperature and cold storage stresses, compared to fermented skim milk without the addition of WPI. Moreover, treatment with the probiotic beverages prevented weight loss and intestinal damages in mice receiving 5-FU. All symptoms of mucositis were drastically reduced by the consumption of developed probiotic fermented milks.ConclusionThis study evidenced the protective effect of selected strains of both lactic and propionic acid bacteria, in the context of induced mucositis. It confirmed that selected strains might be used bot as starter and probiotic. It opens new avenues for the development of fermented functional foods for target populations

Fermented milks, using Lactobacillus casei or Propionibacterium freudenreichii, prevent mucositis, a side effect of chemotherapy, in mice

Mucositis, a common and often debilitating side effect of cancer chemotherapy, is a clinically important gastrointestinal inflammatory disease. It consists in a painful inflammation and ulceration of the mucosa lining the digestive tract, which may compromise proper nutrition of the patient, as well as termination of the treatment. It thus increases mortality and morbidity and contributes to rising health care costs. Its treatment is mainly supportive and often fails to relieve symptoms. Several studies indicate a role of the microbiota and suggest a healing effect of probiotic ingestion. Probiotic bacteria may maintain homeostasis and reduce side effects of chemotherapy toxicity. A major limit to probiotic bacteria anti-inflammatory effect is their susceptibility to digestive stresses and several studies indicate the key protective role of food matrices in increasing probiotics’ effect via the protection of bacteria against digestive constraints. In this study, we investigated the role of fermented milk, using Lactobacillus casei or Propionibacterium freudenreichii as probiotic starter, and of its fortification via addition of whey proteins.Matériels et Méthodes/Materials and methodsWe evaluated the role of whey protein isolate (WPI), when added to skim milk fermented by L. casei BL23 or by P. freudenreichii 138, as a protective matrix against in vitro stress challenges. In addition, we investigated in vivo the therapeutic effect of these fermented beverages in a murine model of mucositis induced by 5-Fluorouracil (5-FU). The monitored outcomes included weight loss, ileum histopathological score, villus height, crypt depth and number of mucus-producing goblet cells.Résultats principaux/Main ResultsThis study demonstrated that milk supplementation with 30% (w/v) of WPI increases the survival rate of both strains when challenged with acid, bile salts, high temperature and cold storage stresses, compared to fermented skim milk without the addition of WPI. Moreover, treatment with the probiotic beverages prevented weight loss and intestinal damages in mice receiving 5-FU. All symptoms of mucositis were drastically reduced by the consumption of developed probiotic fermented milks.ConclusionThis study evidenced the protective effect of selected strains of both lactic and propionic acid bacteria, in the context of induced mucositis. It confirmed that selected strains might be used bot as starter and probiotic. It opens new avenues for the development of fermented functional foods for target populations

Fermented milks, using Lactobacillus casei or Propionibacterium freudenreichii, prevent mucositis, a side effect of chemotherapy, in mice

Mucositis, a common and often debilitating side effect of cancer chemotherapy, is a clinically important gastrointestinal inflammatory disease. It consists in a painful inflammation and ulceration of the mucosa lining the digestive tract, which may compromise proper nutrition of the patient, as well as termination of the treatment. It thus increases mortality and morbidity and contributes to rising health care costs. Its treatment is mainly supportive and often fails to relieve symptoms. Several studies indicate a role of the microbiota and suggest a healing effect of probiotic ingestion. Probiotic bacteria may maintain homeostasis and reduce side effects of chemotherapy toxicity. A major limit to probiotic bacteria anti-inflammatory effect is their susceptibility to digestive stresses and several studies indicate the key protective role of food matrices in increasing probiotics’ effect via the protection of bacteria against digestive constraints. In this study, we investigated the role of fermented milk, using Lactobacillus casei or Propionibacterium freudenreichii as probiotic starter, and of its fortification via addition of whey proteins.Matériels et Méthodes/Materials and methodsWe evaluated the role of whey protein isolate (WPI), when added to skim milk fermented by L. casei BL23 or by P. freudenreichii 138, as a protective matrix against in vitro stress challenges. In addition, we investigated in vivo the therapeutic effect of these fermented beverages in a murine model of mucositis induced by 5-Fluorouracil (5-FU). The monitored outcomes included weight loss, ileum histopathological score, villus height, crypt depth and number of mucus-producing goblet cells.Résultats principaux/Main ResultsThis study demonstrated that milk supplementation with 30% (w/v) of WPI increases the survival rate of both strains when challenged with acid, bile salts, high temperature and cold storage stresses, compared to fermented skim milk without the addition of WPI. Moreover, treatment with the probiotic beverages prevented weight loss and intestinal damages in mice receiving 5-FU. All symptoms of mucositis were drastically reduced by the consumption of developed probiotic fermented milks.ConclusionThis study evidenced the protective effect of selected strains of both lactic and propionic acid bacteria, in the context of induced mucositis. It confirmed that selected strains might be used bot as starter and probiotic. It opens new avenues for the development of fermented functional foods for target populations

Probiotic Propionibacterium freudenreichii requires SlpB protein to mitigate mucositis induced by chemotherapy

Propionibacterium freudenreichii CIRM-BIA 129 (P. freudenreichii wild type, WT) is a probiotic bacterium, which exerts immunomodulatory effects. This strain possesses extractable surface proteins, including SlpB, which are involved in antiinflammatory effect and in adhesion to epithelial cells. We decided to investigate the impact of slpB gene mutation on immunomodulation in vitro and in vivo. In an in vitro assay, P. freudenreichii WT reduced expression of IL-8 (p<0.0001) and TNF-α (p<0.0001) cytokines in LPS-stimulated HT-29 cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. Subsequently, both strains were investigated in vivo in a 5-FU-induced mucositis mice model. Mucositis is a common side effect of cytotoxic chemotherapy with 5-FU, characterized by mucosal injury, inflammation, diarrhea, and weight loss. The WT strain prevented weight loss, reduced inflammation and consequently histopathological scores. Furthermore, it regulated key markers, including Claudin-1 (cld1, p<0.0005) and IL-17a (Il17a, p<0.0001) genes, as well as IL-12 (p<0.0001) and IL-1β (p<0.0429) cytokines levels. Mutant strain displayed opposite regulatory effect on cld1 expression and on IL-12 levels. This work emphasizes the importance of SlpB in P. freudenreichii ability to reduce mucositis inflammation. It opens perspectives for the development of probiotic products to decrease side effects of chemotherapy using GRAS bacteria with immunomodulatory surface protein properties