RESUMEN
Influenza A annually infects 5-10% of the world's human population resulting in one million deaths. Influenza causes annual epidemics and reinfects previously exposed individuals because of antigenic drift in the glycoprotein hemagglutinin. Due to antigenic drift, the immune system is simultaneously exposed to novel and conserved parts of the influenza virus via vaccination and/or infection throughout life. Preexisting immunity has long been known to augment subsequent hemagglutination inhibitory antibody (hAb) responses. However, the preexisting immunological contributors that influence hAb responses are not understood. Therefore, we adapted and developed sequential infection and immunization mouse models using drifted influenza strains to show that MHC Class II haplotype and T-cell reactivity influences subsequent hAb responses. We found that CB6F1 mice infected with A/CA followed by immunization with A/PR8 have increased hAb responses to A/PR8 compared to C57BL/6 mice. Increased hAb responses in CB6F1 mice were CD4+ T-cell and B-cell dependent and corresponded to increased germinal center A/PR8-specific B and T-follicular helper cells. These results suggest conserved MHC Class II restricted epitopes within HA are essential for B cells to respond to drifting influenza and could be leveraged to boost hAb responses.
Asunto(s)
Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Inmunización , Memoria Inmunológica , Virus de la Influenza A/inmunología , Animales , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Femenino , RatonesRESUMEN
The involvement of innate receptors that recognize pathogen- and danger-associated molecular patterns is critical to programming an effective adaptive immune response to vaccination. The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) synergizes with the squalene oil-in-water emulsion (SE) formulation to induce strong adaptive responses. Although TLR4 signaling through MyD88 and TIR domain-containing adapter inducing IFN-ß are essential for GLA-SE activity, the mechanisms underlying the synergistic activity of GLA and SE are not fully understood. In this article, we demonstrate that the inflammasome activation and the subsequent release of IL-1ß are central effectors of the action of GLA-SE, as infiltration of innate cells into the draining lymph nodes and production of IFN-γ are reduced in ASC-/- animals. Importantly, the early proliferation of Ag-specific CD4+ T cells was completely ablated after immunization in ASC-/- animals. Moreover, numbers of Ag-specific CD4+ T and B cells as well as production of IFN-γ, TNF-α, and IL-2 and Ab titers were considerably reduced in ASC-/-, NLRP3-/-, and IL-1R-/- mice compared with wild-type mice and were completely ablated in TLR4-/- animals. Also, extracellular ATP, a known trigger of the inflammasome, augments Ag-specific CD4+ T cell responses, as hydrolyzing it with apyrase diminished adaptive responses induced by GLA-SE. These data thus demonstrate that GLA-SE adjuvanticity acts through TLR4 signaling and NLRP3 inflammasome activation to promote robust Th1 and B cell responses to vaccine Ags. The findings suggest that engagement of both TLR and inflammasome activators may be a general paradigm for induction of robust CD4 T cell immunity with combination adjuvants such as GLA-SE.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos/inmunología , Linfocitos B/inmunología , Inflamasomas/inmunología , Células TH1/inmunología , Receptor Toll-Like 4/inmunología , Vacunas/inmunología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Femenino , Glucósidos/inmunología , Inmunidad Humoral , Interferón beta/inmunología , Interferón gamma/inmunología , Interleucina-1beta/metabolismo , Interleucina-2/inmunología , Lípido A/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Receptores Tipo I de Interleucina-1/genética , Escualeno/inmunología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/inmunología , VacunaciónRESUMEN
Designing modern vaccine adjuvants depends on understanding the cellular and molecular events that connect innate and adaptive immune responses. The synthetic TLR4 agonist glycopyranosyl lipid adjuvant (GLA) formulated in a squalene-in-water emulsion (GLA-SE) augments both cellular and humoral immune responses to vaccine Ags. This adjuvant is currently included in several vaccines undergoing clinical evaluation including those for tuberculosis, leishmaniasis, and influenza. Delineation of the mechanisms of adjuvant activity will enable more informative evaluation of clinical trials. Early after injection, GLA-SE induces substantially more Ag-specific B cells, higher serum Ab titers, and greater numbers of T follicular helper (TFH) and Th1 cells than alum, the SE alone, or GLA without SE. GLA-SE augments Ag-specific B cell differentiation into germinal center and memory precursor B cells as well as preplasmablasts that rapidly secrete Abs. CD169+ SIGNR1+ subcapsular medullary macrophages are the primary cells to take up GLA-SE after immunization and are critical for the innate immune responses, including rapid IL-18 production, induced by GLA-SE. Depletion of subcapsular macrophages (SCMÑ) or abrogation of IL-18 signaling dramatically impairs the Ag-specific B cell and Ab responses augmented by GLA-SE. Depletion of SCMÑ also drastically reduces the Th1 but not the TFH response. Thus the GLA-SE adjuvant operates through interaction with IL-18-producing SCMÑ for the rapid induction of B cell expansion and differentiation, Ab secretion, and Th1 responses, whereas augmentation of TFH numbers by GLA-SE is independent of SCMÑ.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B/inmunología , Diferenciación Celular/efectos de los fármacos , Glucósidos/farmacología , Interleucina-18/inmunología , Lípido A/farmacología , Ganglios Linfáticos/inmunología , Macrófagos/inmunología , Receptor Toll-Like 4/agonistas , Animales , Linfocitos B/citología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Diferenciación Celular/inmunología , Femenino , Glucósidos/farmacocinética , Interleucina-18/genética , Subunidad alfa del Receptor de Interleucina-18/genética , Subunidad alfa del Receptor de Interleucina-18/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Lípido A/farmacocinética , Ganglios Linfáticos/citología , Macrófagos/citología , Ratones , Ratones Noqueados , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 1 Similar a Ig de Unión al Ácido Siálico/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Células TH1/citología , Células TH1/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T-helper 1 (TH 1) CD4+ T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B-cell deficient mice (µMT-/- ), tetramer-positive CD4+ T cells, markers of memory "precursor" effector cells (MPECs), and T-cell adoptive transfers and demonstrated that the early antigen-specific cytokine-producing TH 1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory TH 1 response in µMT-/- mice. We further show that antigen-presentation by B cells is necessary for their role in MPEC generation using B-cell adoptive transfers from wt or MHC class II knock-out mice into µMT-/- mice. Our study challenges the view that B-cell deficiency exclusively alters the TH 1 response at memory time-points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring TH 1 cell-mediated immunity.
Asunto(s)
Presentación de Antígeno , Linfocitos B/fisiología , Factores Inmunológicos/inmunología , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/inmunología , Traslado Adoptivo , Animales , Linfocitos B/trasplante , Diferenciación Celular , Células Cultivadas , Humanos , Cadenas mu de Inmunoglobulina/genética , Memoria Inmunológica , Activación de Linfocitos , Ratones , Ratones Noqueados , Subgrupos de Linfocitos T/trasplante , Receptor Toll-Like 4/agonistas , Tuberculosis/prevención & controlRESUMEN
The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) is a potent Th1-response-inducing adjuvant when formulated in a squalene oil-in-water emulsion (SE). While the innate signals triggered by TLR4 engagement are well studied, the contribution of SE remains unclear. To better understand the effect of SE on the adjuvant properties of GLA-SE, we compared the innate and adaptive immune responses elicited by immunization with different formulations: GLA without oil, SE alone or the combination, GLA-SE, in mice. Within the innate response to adjuvants, only GLA-SE displayed features of inflammasome activation, evidenced by early IL-18 secretion and IFN-γ production in memory CD8(+) T cells and neutrophils. Such early IFN-γ production was ablated in caspase-1/11(-/-) mice and in IL-18R1(-/-) mice. Furthermore, caspase-1/11 and IL-18 were also required for full Th1 CD4(+) T-cell induction via GLA-SE. Thus, we demonstrate that IL-18 and caspase-1/11 are components of the response to immunization with the TLR4 agonist/squalene oil-in-water based adjuvant, GLA-SE, providing implications for other adjuvants that combine oils with TLR agonists.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Caspasa 1/inmunología , Caspasas/inmunología , Interferón gamma/inmunología , Interleucina-18/inmunología , Escualeno/administración & dosificación , Receptor Toll-Like 4/agonistas , Inmunidad Adaptativa/efectos de los fármacos , Adyuvantes Inmunológicos/síntesis química , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Caspasa 1/genética , Caspasas/genética , Caspasas Iniciadoras , Emulsiones , Femenino , Expresión Génica , Inmunidad Innata/efectos de los fármacos , Inmunización , Memoria Inmunológica , Inflamasomas/efectos de los fármacos , Interferón gamma/biosíntesis , Interleucina-18/biosíntesis , Lípidos/administración & dosificación , Lípidos/síntesis química , Lípidos/inmunología , Ratones , Ratones Noqueados , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Receptores de Interleucina-18/genética , Receptores de Interleucina-18/inmunología , Escualeno/química , Escualeno/inmunología , Células TH1/citología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Melioidosis is an often fatal infectious disease affecting humans and animals in the tropics. Only sporadic cases have been reported from Africa and the Indian Ocean region. We describe 2 confirmed autochthonous cases of human melioidosis in Madagascar, both from novel genotypes of Burkholderia pseudomallei.
Asunto(s)
Melioidosis/epidemiología , Antibacterianos/uso terapéutico , Resultado Fatal , Humanos , Madagascar/epidemiología , Masculino , Melioidosis/tratamiento farmacológico , Persona de Mediana EdadRESUMEN
We examined the feasibility of using DNA extracted from stained sputum smears for the detection of rifampin and isoniazid resistance with the commercial MTBDRplus assay from Hain Lifescience GmbH, Nehren, Germany. Overall sensitivity was initially low (70.0%) but increased to 96.7% when a multiplex PCR preamplification step was added. We then tested stored Mycobacterium tuberculosis-positive stained smears prepared from 297 patients' sputum samples. Species identification and drug susceptibility testing (DST) had been performed at the Institut Pasteur de Madagascar. Overall, the performance of the MTBDRplus assay applied to slide DNA was similar to that obtained in other studies with DNA extracted from clinical specimens. With the ready availability of stained smears in routine diagnostic laboratories and their easy transport and storage at room temperature, this approach should be useful for optimizing the treatment of multidrug-resistant tuberculosis and for conducting resistance surveys aimed at identifying hot-spot regions and breaking chains of transmission.
Asunto(s)
Farmacorresistencia Bacteriana , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Manejo de Especímenes/métodos , Esputo/microbiología , Antituberculosos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Sensibilidad y EspecificidadRESUMEN
It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis (Mtb). This astounding statistic, in combination with costly and lengthy treatment regimens make the development of therapeutic vaccines paramount for controlling the global burden of tuberculosis. Unlike prophylactic vaccination, therapeutic immunization relies on the natural pulmonary infection with Mtb as the mucosal prime that directs boost responses back to the lung. The purpose of this work was to determine the protection and safety profile over time following therapeutic administration of our lead Mtb vaccine candidate, ID93 with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE)), in combination with rifampicin, isoniazid, and pyrazinamide (RHZ) drug treatment. We assessed the host inflammatory immune responses and lung pathology 7â»22 weeks post infection, and determined the therapeutic efficacy of combined treatment by enumeration of the bacterial load and survival in the SWR/J mouse model. We show that drug treatment alone, or with immunotherapy, tempered the inflammatory responses measured in brochoalveolar lavage fluid and plasma compared to untreated cohorts. RHZ combined with therapeutic immunizations significantly enhanced TH1-type cytokine responses in the lung over time, corresponding to decreased pulmonary pathology evidenced by a significant decrease in the percentage of lung lesions and destructive lung inflammation. These data suggest that bacterial burden assessment alone may miss important correlates of lung architecture that directly contribute to therapeutic vaccine efficacy in the preclinical mouse model. We also confirmed our previous finding that in combination with antibiotics therapeutic immunizations provide an additive survival advantage. Moreover, therapeutic immunizations with ID93/GLA-SE induced differential T cell immune responses over the course of infection that correlated with periods of enhanced bacterial control over that of drug treatment alone. Here we advance the immunotherapy model and investigate reliable correlates of protection and Mtb control.
RESUMEN
BACKGROUND: Adjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE). MATERIALS AND METHODS: In this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations. RESULTS: We identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies. CONCLUSION: These efforts will aid in the development of a platform formulation for use with other similar vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Emulsiones/química , Nanopartículas/química , Temperatura , Vacunas contra la Tuberculosis/inmunología , Animales , Formación de Anticuerpos , Química Farmacéutica , Dispersión Dinámica de Luz , Excipientes , Femenino , Liofilización , Concentración de Iones de Hidrógeno , Inmunidad Celular , Lípidos/química , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/inmunología , Nefelometría y Turbidimetría , Tamaño de la Partícula , Tuberculosis/inmunología , Tuberculosis/patologíaRESUMEN
The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant formulated in a stable emulsion (GLA-SE) promotes strong TH1 and balanced IgG1/IgG2 responses to protein vaccine antigens. This enhanced immunity is sufficient to provide protection against many diseases including tuberculosis and leishmaniasis. To better characterize the adjuvant action it is important to understand how the different cytokines and transcription factors contribute to the initiation of immunity. In the present study using T-bet-/- and IL-12-/- mice and a blocking anti-IFNαR1 monoclonal antibody, we define mechanisms of adjuvant activity of GLA-SE. In accordance with previous studies of TLR4 agonist based adjuvants, we found that TH1 induction via GLA-SE was completely dependent upon T-bet, a key transcription factor for IFNγ production and TH1 differentiation. Consistent with this, deficiency of IL-12, a cytokine canonical to TH1 induction, ablated TH1 induction via GLA-SE. Finally we demonstrate that the innate immune response to GLA-SE, including rapid IFNγ production by memory CD8+ T cells and NK cells, was contingent on type I interferon, a cytokine group whose association with TH1 induction is contextual, and that they contributed to the adjuvant activity of GLA-SE.