Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responsesFil: Hiriart, Yanina. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Rossi, Andrés Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Biedma, Marina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Errea, Agustina Juliana. Universidad Nacional de La Plata; Argentina. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Moreno, Griselda Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Cayet, D.. Universidad Nacional de La Plata; ArgentinaFil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Blancá, Bruno Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Sirard, J.C.. Centre National de la Recherche Scientifique; FranciaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentin

The scavenger receptors SRA-1 and SREC-I cooperate with TLR2 in the recognition of the hepatitis C virus non-structural protein 3 by dendritic cells

Backgrounds & AimsThe hepatitis C virus NS3 protein is taken up by myeloid cells in a TLR2-independent manner and activates myeloid cells via TLR2. This study aimed to identify the endocytic receptor(s) involved in the uptake of NS3 by myeloid cells and its relation with TLR2. Methods Inhibitors and transfected cells were used to identify the nature of the NS3-binding receptors expressed by myeloid cells. The cooperation between scavenger receptors (SRs) and TLR2 in the NS3-mediated activation of myeloid cells was evaluated using inhibitors, cells from TLR2−/− mice, and confocal microscopy. The involvement of SRs in NS3 cross-presentation was evaluated in vitro using an NS3-specific human T-cell clone. Results We observed that SRs are the main binding structures for NS3 on myeloid cells and identified the SRs SRA-1 and SREC-I as endocytic receptors for NS3. Moreover, both SRs and TLR2 cooperate in NS3-induced myeloid cell activation. Conclusion This study highlights a central role for SRs in NS3 uptake and cross-presentation, and demonstrates a tightly orchestrated cooperation between signalling and endocytic innate receptors in NS3 recognition

Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responsesInstituto de Estudios Inmunológicos y Fisiopatológico

Airway structural cells regulate TLR5-mediated mucosal adjuvant activity

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin’s mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.Fil: Van Maele, Laurye. Institut Pasteur de Lille. Lille; Francia. Univ Lille Nord de France. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Fougeron, Delphine. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Janot, Laurent. University of Orléans. Orléans; Francia. Institut de Transgenose. Orleans; FranciaFil: Didierlaurent, A.. Imperial College of London. Londres; Reino UnidoFil: Cayet, D.. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Tabareau, J.. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Corvo Chamaillard, S.. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Boulenoir, S.. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Jeffs, S. Imperial College of London. Londres; Reino UnidoFil: Vande Walle, L. Department of Medical Protein Research. Ghent; Bélgica. University of Ghent; BélgicaFil: Lamkanfi, M.. Department of Medical Protein Research. Ghent; Bélgica. University of Ghent; BélgicaFil: Lemoine, Y.. Univ Lille Nord de France. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Institut Pasteur de Lille. Lille; FranciaFil: Erard, F.. Institut de Transgenose. Orleans; Francia. University of Orléans. Orléans; FranciaFil: Hot, D.. Univ Lille Nord de France. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Institut Pasteur de Lille. Lille; FranciaFil: Hussell, Tracy. Imperial College of London. Londres; Reino Unido. University of Manchester; Reino UnidoFil: Ryffel, B.. Institut de Transgenose. Orleans; Francia. University of Orléans. Orléans; FranciaFil: Benecke, Arndt G.. Institut des Hautes Études Scientifiques and Centre National de la Recherche Scientifique; FranciaFil: Sirard, J.C.. Univ Lille Nord de France. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Institut Pasteur de Lille. Lille; Franci

Live attenuated Salmonella: a paradigm of mucosal vaccines.

Two key steps control immune responses in mucosal tissues: the sampling and transepithelial transport of antigens, and their targeting into professional antigen-presenting cells in mucosa-associated lymphoid tissue. Live Salmonella bacteria use strategies that allow them to cross the epithelial barrier of the gut, to survive in antigen-presenting cells where bacterial antigens are processed and presented to the immune cells, and to express adjuvant activity that prevents induction of oral tolerance. Two Salmonella serovars have been used as vaccines or vectors, S. typhimurium in mice and S. typhi in humans. S. typhimurium causes gastroenteritis in a broad host range, including humans, while S. typhi infection is restricted to humans. Attenuated S. typhimurium has been used successfully in mice to induce systemic and mucosal responses against more than 60 heterologous antigens. This review aims to revisit S. typhimurium-based vaccination, as an alternative to S. typhi, with special emphasis on the molecular pathogenesis of S. typhimurium and the host response. We then discuss how such knowledge constitutes the basis for the rational design of novel live mucosal vaccines

Dendritic cells: the host Achille's heel for mucosal pathogens?

Mucosal surfaces represent the main sites of interaction with environmental microorganisms and antigens. Sentinel cells, including epithelial cells and dendritic cells (DCs), continuously sense the environment and coordinate defenses for the protection of mucosal tissues. DCs play a central role in the control of adaptive immune responses owing to their capacity to internalize foreign materials, to migrate into lymph nodes and to present antigens to naive lymphocytes. Some pathogenic microorganisms trigger epithelial responses that result in the recruitment of DCs. These pathogens hijack the recruited DCs to enable them to infect the host, escape the host's defense mechanisms and establish niches at remote sites

Attenuated poxviruses expressing a synthetic HIV protein stimulate HLA-A2-restricted cytotoxic T-cell responses.

Efficient HIV vaccines have to trigger cell-mediated immunity directed against various viral antigens. However little is known about the breadth of the response induced by vaccines carrying multiple proteins. Here, we report on the immunogenicity of a construct harbouring a fusion of the HIV-1 IIIB gag, pol and nef genes (gpn) designed for optimal safety and equimolar expression of the HIV proteins. The attenuated poxviruses, MVA and NYVAC, harbouring the gpn construct, induced potent immune responses in conventional mice characterised by stimulation of Gpn-specific IFN-gamma-producing cells and cytotoxic T cells. In HLA-A2 transgenic mice, recombinant MVA elicited cytotoxic responses against epitopes recognised in most HLA-A2+ HIV-1-infected individuals. We also found that the MVA vaccine triggered the in vitro expansion of peripheral blood cells isolated from a HIV-1-seropositive patient and with similar specificity as found in immunised HLA-A2 transgenic mice. In conclusion, the synthetic HIV polyantigen Gpn delivered by MVA is immunogenic, efficiently processed and presented by human MHC class I molecules

How the gut links innate and adaptive immunity.

Mucosal surfaces represent the main sites in which environmental microorganisms and antigens interact with the host. Sentinel cells, including epithelial cells, lumenal macrophages, and intraepithelial dendritic cells, continuously sense the environment and coordinate defenses for the protection of mucosal tissues. The mucosal epithelial cells are crucial actors in coordinating defenses. They sense the outside world and respond to environmental signals by releasing chemokines and cytokines that recruit inflammatory and immune cells to control potential infectious agents and to attract cells able to trigger immune responses. Among immune cells, dendritic cells (DC) play a key role in controlling adaptive immune responses, due to their capacity to internalize foreign materials and to present antigens to naive T and B lymphocytes, locally or in draining organized lymphoid tissues. Immune cells recruited in epithelial tissues can, in turn, act upon the epithelial cells and change their phenotype in a process referred to as epithelial metaplasia

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as indicated by sCRilecs-seq.

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the β-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by a reduced availability of cell wall precursors at the site of cell wall synthesis due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier that is responsible for transporting these precursors across the cell membrane. The data suggest that, whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections

Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq.

Streptococcus pneumoniae is an opportunistic human pathogen that causes invasive diseases, including pneumonia, with greater health risks upon influenza A virus (IAV) co-infection. To facilitate pathogenesis studies in vivo, we developed an inducible CRISPR interference system that enables genome-wide fitness testing in one sequencing step (CRISPRi-seq). We applied CRISPRi-seq to assess bottlenecks and identify pneumococcal genes important in a murine pneumonia model. A critical bottleneck occurs at 48 h with few bacteria causing systemic infection. This bottleneck is not present during IAV superinfection, facilitating identification of pneumococcal pathogenesis-related genes. Top in vivo essential genes included purA, encoding adenylsuccinate synthetase, and the cps operon required for capsule production. Surprisingly, CRISPRi-seq indicated no fitness-related role for pneumolysin during superinfection. Interestingly, although metK (encoding S-adenosylmethionine synthetase) was essential in vitro, it was dispensable in vivo. This highlights advantages of CRISPRi-seq over transposon-based genetic screens, as all genes, including essential genes, can be tested for pathogenesis potential