Neonate Intestinal Immune Response to CpG Oligodeoxynucleotide Stimulation

Background: The development of mucosal vaccines is crucial to efficiently control infectious agents for which mucosae are the primary site of entry. Major drawbacks of these protective strategies are the lack of effective mucosal adjuvant. Synthetic oligodeoxynucleotides that contain several unmethylated cytosine-guanine dinucleotide (CpG-ODN) motifs are now recognized as promising adjuvants displaying mucosal adjuvant activity through direct activation of TLR9-expressing cells. However, little is known about the efficacy of these molecules in stimulating the intestinal immune system in neonates. Methodology/Principal Findings: First, newborn mice received CpG-ODN orally, and the intestinal cytokine and chemokine response was measured. We observed that oral administration of CpG-ODN induces CXC and CC chemokine responses and a cellular infiltration in the intestine of neonates as detected by immunohistochemistry. We next compared the efficiency of the oral route to intraperitoneal administration in stimulating the intestinal immune responses of both adults and neonates. Neonates were more responsive to TLR9-stimulation than adults whatever the CpG-ODN administration route. Their intestinal epithelial cells (IECs) indirectly responded to TLR9 stimulation and contributed to the CXC chemokine response, whereas other TLR9-bearing cells of the lamina-propria produced CC chemokines and Th1-type cytokines. Moreover, we showed that the intestine of adult exhibited a significantly higher level of IL10 at homeostasis than neonates, which might be responsible for the unresponsiveness to TLR9-stimulation, as confirmed by our findings in IL10-deficient mice. Conclusions/Significance: This is the first report that deciphers the role played by CpG-ODN in the intestine of neonates. This work clearly demonstrates that an intraperitoneal administration of CpG-ODN is more efficient in neonates than in adults to stimulate an intestinal chemokine response due to their lower IL-10 intestinal level. In addition we report the efficiency of the oral route at inducing intestinal chemokine responses in neonate that might be taken into consideration for further vaccine development against neonatal diseases

Rye cell wall β-glucosidase: subcloning, expression and purification of recombinant protein from E.coli

Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly

Evaluation of HIV-1 vaccine strategies based on the use of SigA as mucosal addressing molecule

Les SIgA possèdent la capacité de pouvoir adhérer spécifiquement à la membrane apicale des cellules M présentes au niveau des muqueuses monostratifiées. La capacité des cellules M à transporter les SIgA de la lumière intestinale jusqu'au GALT par un mécanisme de transcytose inverse a également été décrite. J'ai donc souhaité évaluer la capacité des SIgA à transporter efficacement un antigène vaccinal, à travers la barrière épithéliale par l'intermédiaire des cellules M vers les DCs présentes dans le MALT. Le mécanisme exact et notamment la structure moléculaire du récepteur permettant la transcytose inverse des IgA n'a pas été identifiée. Il m’a donc paru intéressant d'améliorer la compréhension des mécanismes physiologiques impliqués dans le transport d'une SIgA de la lumière intestinale jusqu’aux cellules immunitaires sous-muqueuses. Cette étude a permis de démontrer le rôle majeur de deux nouveaux récepteurs présents à la surface des cellules M, la dectine-1 et le siglec-5, dans l'activité physiologique rétrograde des SIgA. Cette étude a permis d'identifier les domaines des SIgA impliqués dans ce mécanisme. J'ai ensuite utilisé les SIgA comme vecteur vaccinal permettant le ciblage des cellules M. Les applications de cette approche à la vaccination par voie orale et nasale sont décrites dans la publication 4 en cours de rédaction. Durant ma thèse, j'ai pu démontrer que la transcytose inverse des SIgA est un mécanisme physiologique dépendant par exemple de récepteurs aux sucres. J'ai pu également démontrer que leur utilisation dans des approches de vaccination muqueuse peuvent être une voie très prometteuse notamment contre le VIH ou d'autres pathogènes muqueuxSecretory IgA (SIgA) are the main effectors of the mucosal immune response. More, SIgA have the capacity to adhere to the apical membrane of M cells present in the intestinal and nasal mucosa. After binding to M cells, SIgA are transported from the intestinal lumen to the GALT by a reverse transcytosis mechanism. In this work, I have assessed the capacity of SIgA to effectively deliver a vaccine antigen through the epithelial barrier via M cells to sub-mucosal dendritic cells (DCs). Precise mechanisms and the IgA-specific receptor(s) for reverse transcytosis have not yet been identified. In this work, I identified the receptors involved in SIgA reverse transcytosis. Both dectin-1 and siglec-5 allow the transport of the Cα1 domain of SIgA by murine an human M cells in vitro and also in vivo. This work is currently undergoing to immunity (publication 1) and should also be patented. Next, I tried to use the reverse transcytosis mechanism mediated by M cells to efficiently deliver an HIV-1 antigen by mucosal routes. We applied results obtained using SIgA as a vaccine vector for M cells targeting. This approach should help to protect antigen in the mucosal environment. Applications of this approach to oral and nasal immunisation are described in the incomplete publication 4. During any PhD, I was able to demonstrate that SIgA reverse transcytosis is a physiological mechanism depending on sugar receptors. I was also able to demonstrate that their use could be a very promising vaccine approach especially for mucosa] diseases or pathogens as HI

Evaluation de stratégies vaccinales anti-VIH-1 basées sur l'utilisation de SIgA comme molécules d'adressage muqueux

Les SIgA possèdent la capacité de pouvoir adhérer spécifiquement à la membrane apicale des cellules M présentes au niveau des muqueuses monostratifiées. La capacité des cellules M à transporter les SIgA de la lumière intestinale jusqu'au GALT par un mécanisme de transcytose inverse a également été décrite. J'ai donc souhaité évaluer la capacité des SIgA à transporter efficacement un antigène vaccinal, à travers la barrière épithéliale par l'intermédiaire des cellules M vers les DCs présentes dans le MALT. Le mécanisme exact et notamment la structure moléculaire du récepteur permettant la transcytose inverse des IgA n'a pas été identifiée. Il m a donc paru intéressant d'améliorer la compréhension des mécanismes physiologiques impliqués dans le transport d'une SIgA de la lumière intestinale jusqu aux cellules immunitaires sous-muqueuses. Cette étude a permis de démontrer le rôle majeur de deux nouveaux récepteurs présents à la surface des cellules M, la dectine-1 et le siglec-5, dans l'activité physiologique rétrograde des SIgA. Cette étude a permis d'identifier les domaines des SIgA impliqués dans ce mécanisme. J'ai ensuite utilisé les SIgA comme vecteur vaccinal permettant le ciblage des cellules M. Les applications de cette approche à la vaccination par voie orale et nasale sont décrites dans la publication 4 en cours de rédaction. Durant ma thèse, j'ai pu démontrer que la transcytose inverse des SIgA est un mécanisme physiologique dépendant par exemple de récepteurs aux sucres. J'ai pu également démontrer que leur utilisation dans des approches de vaccination muqueuse peuvent être une voie très prometteuse notamment contre le VIH ou d'autres pathogènes muqueuxSecretory IgA (SIgA) are the main effectors of the mucosal immune response. More, SIgA have the capacity to adhere to the apical membrane of M cells present in the intestinal and nasal mucosa. After binding to M cells, SIgA are transported from the intestinal lumen to the GALT by a reverse transcytosis mechanism. In this work, I have assessed the capacity of SIgA to effectively deliver a vaccine antigen through the epithelial barrier via M cells to sub-mucosal dendritic cells (DCs). Precise mechanisms and the IgA-specific receptor(s) for reverse transcytosis have not yet been identified. In this work, I identified the receptors involved in SIgA reverse transcytosis. Both dectin-1 and siglec-5 allow the transport of the Ca1 domain of SIgA by murine an human M cells in vitro and also in vivo. This work is currently undergoing to immunity (publication 1) and should also be patented. Next, I tried to use the reverse transcytosis mechanism mediated by M cells to efficiently deliver an HIV-1 antigen by mucosal routes. We applied results obtained using SIgA as a vaccine vector for M cells targeting. This approach should help to protect antigen in the mucosal environment. Applications of this approach to oral and nasal immunisation are described in the incomplete publication 4. During any PhD, I was able to demonstrate that SIgA reverse transcytosis is a physiological mechanism depending on sugar receptors. I was also able to demonstrate that their use could be a very promising vaccine approach especially for mucosa] diseases or pathogens as HIVST ETIENNE-Bib. électronique (422189901) / SudocSudocFranceF

Rôle et ciblage de l’intégrine α4β7 dans la physiopathologie des MICI et de l’infection par le VIH

Les intégrines forment une grande famille de molécules impliquées dans de nombreux processus biologiques. Elles orchestrent les mécanismes d’adhésion entre cellules et matrice extracellulaire, dès le développement embryonnaire et pendant toute la vie adulte. Ces molécules hétérodimériques participent à beaucoup de fonctions biologiques comme la migration, la prolifération, la différenciation et la survie cellulaire. Elles sont également impliquées dans différentes pathologies humaines dont les maladies thrombotiques, l’inflammation, les cancers, la fibrose et les infections. Leur exposition à la surface cellulaire en fait des cibles thérapeutiques séduisantes, et, de fait, plusieurs molécules ciblant les intégrines ont prouvé leur efficacité en thérapeutique humaine. Dans cette revue, nous nous focaliserons sur l’intégrine α4β7, particulièrement intéressante par son implication dans différentes pathologies touchant les muqueuses, comme les maladies inflammatoires chroniques de l’intestin (MICI) et, plus récemment, l’infection par le virus VIH

Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells in gut mucosa

International audienceMucosal immunization is designed to induce strong immune responses at portal of pathogen entry. Unfortunately, mechanisms underlying the fate of the vaccine vector co-administered with antigens are still partially uncovered and limit further development of mucosal vaccines. Hence, poly(lactic acid) (PLA) nanoparticles being a versatile vaccine vehicle, we have analyzed the fate of these PLA nanoparticles during their uptake at intestinal mucosa] sites, both in vivo and ex vivo, to decipher the mechanisms involved during this process. We first designed specific fluorescent PLA nanoparticles exhibiting strong colloidal stability after encapsulation of either 6-coumarin or CellTrace BODIPY (R) before monitoring their transport through mucosa in the mouse ligated ileal loop model. The journey of the particles appears to follow a three-step process. Most particles are first entrapped in the mucus. Then, crossing of the epithelial barrier takes place exclusively through M-cells, leading to an accumulation in Peyer's patches (PP). Lastly, we noticed specific interaction of these PLA nanoparticles with underlying B cells and dendritic cells (DCs) of PP. Furthermore, we could document that DCs engulfing some nanoparticles could exhibit a TLR8+ specific expression. Specific targeting of these two cell types strongly supports the use of PLA nanoparticles as a vaccine delivery system for oral use. Indeed, following oral gavage of mice with PLA nanoparticles, we were able to observe the same biodistribution patterns, indicating that these nanoparticles specifically reach immune target required for oral immunization

Recent progress in HIV vaccines inducing mucosal immune responses

International audienceIn spite of several attempts over many years at developing a HIV vaccine based on classical strategies, none has convincingly succeeded to date. As HIV is transmitted primarily by the mucosal route, particularly through sexual intercourse, understanding antiviral immunity at mucosal sites is of major importance. An ideal vaccine should elicit HIV-specific antibodies and mucosal CD8^+ cytotoxic T-lymphocyte (CTL) as a first line of defense at a very early stage of HIV infection, before the virus can disseminate into the secondary lymphoid organs in mucosal and systemic tissues. A primary focus of HIV preventive vaccine research is therefore the induction of protective immune responses in these crucial early stages of HIV infection. Numerous approaches are being studied in the field, including building upon the recent RV144 clinical trial. In this article, we will review current strategies and briefly discuss the use of adjuvants in designing HIV vaccines that induce mucosal immune responses

Antiviral Activities of HIV-1-Specific Human Broadly Neutralizing Antibodies Are Isotype-Dependent

Broadly neutralizing antibodies (bNAbs) offer promising opportunities for preventing HIV-1 infection. The protection mechanisms of bNAbs involve the Fc domain, as well as their Fab counterpart. Here, different bNAb isotypes including IgG1, IgA1, IgA2, and IgA122 (IgA2 with the hinge of IgA1) were generated and then produced in CHO cells. Their ability to neutralize pseudovirus and primary HIV-1 isolates were measured, as well as their potential ADCC-like activity using a newly developed assay. In our work, gp41-specific IgA seems to be more efficient than IgG1 in inducing ADCC-like activity, but not in its virus neutralization effect. We show that either gp120-specific IgA or IgG1 isotypes are both efficient in neutralizing different viral strains. In contrast, gp120-specific IgG1 was a better ADCC-like inducer than IgA isotypes. These results provide new insights into the neutralization and ADCC-like activity of different bNAbs that might be taken into consideration when searching for new treatments or antibody-based vaccines