RESUMO
Thirty years of intensive research have significantly contributed to our understanding of Helicobacter pylori biology and pathogenesis. However, the lack of convenient genetic tools, in particular the limited effectiveness of available reporter systems, has notably limited the toolbox for fundamental and applied studies. Here, we report the construction of a bioluminescent H. pylori reporter system based on the Photorhabdus luminescens luxCDABE cassette. The system is constituted of a promoterless lux acceptor strain in which promoters and sequences of interest can be conveniently introduced by double homologous recombination of a suicide transformation vector. We validate the robustness of this new lux reporter system in noninvasive in vivo monitoring of dynamic transcriptional responses of inducible as well as repressible promoters and demonstrate its suitability for the implementation of genetic screens in H. pylori.
Assuntos
Perfilação da Expressão Gênica/métodos , Genes Reporter , Helicobacter pylori/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Vetores Genéticos , Humanos , Medições Luminescentes , Dados de Sequência Molecular , Photorhabdus/enzimologia , Photorhabdus/genética , Recombinação Genética , Análise de Sequência de DNARESUMO
The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas contra Influenza/imunologia , Interferon-alfa/imunologia , Lipopeptídeos/farmacologia , Polissorbatos/farmacologia , Esqualeno/farmacologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Quimiotaxia/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Imunidade Inata/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza B/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Subpopulações de Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Músculos/imunologia , Músculos/metabolismo , Organismos Livres de Patógenos Específicos , Baço/citologia , Baço/imunologia , Baço/metabolismo , Toxoide Tetânico/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Despite the fact that alum and oil-in-water emulsions have been used for decades as human vaccine adjuvants in a large number of individuals, their mechanism of action is not completely understood. It has been reported that these particulate adjuvants act by increasing antigen availability and uptake by immune cells. However, recent work on alum and on the squalene-based emulsion MF59, has demonstrated that besides antigen delivery functions, these classes of adjuvants can also activate innate immunity pathways in vivo, generating an immunocompetent environment at injection site. Interestingly, it has been demonstrated that alum adjuvanticity depends on the activation of a protein complex called NLPR3/inflammasome, which is required for the correct processing of a number of pro-inflammatory cytokines, including IL1beta. More work needs to be performed to investigate if the inflammasome is also required for the activity of MF59 and of other particulate vaccine adjuvants.
