RESUMEN
Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas de Interacción con los Canales Kv/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Factores Estimuladores hacia 5'/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Animales , Inmunoprecipitación de Cromatina , Cisteína Endopeptidasas , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Immunoblotting , Inflamación/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Infection is the predominant cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, very young infants fail to respond optimally to most vaccines currently in use, especially neonates. In 2005, Stanley Plotkin proposed that new delivery systems would spur a new revolution in pediatric vaccinology, just as attenuation, inactivation, cell culture of viruses, genetic engineering, and adjuvantation had done in preceding decades. Recent advances in the field of immunoengineering, which is evolving alongside vaccinology, have begun to increasingly influence vaccine formulation design. Historically, the particulate nature of materials used in many vaccine formulations was empiric, often because of the need to stabilize antigens or reduce endotoxin levels. However, present vaccine delivery systems are rationally engineered to mimic the size, shape, and surface chemistry of pathogens, and are therefore often referred to as "pathogen-like particles". More than a decade from his original assessment, we re-assess Plotkin's prediction. In addition, we highlight how immunoengineering and advanced delivery systems may be uniquely capable of enhancing vaccine responses in vulnerable populations, such as infants. IMPACT: Immunoengineering and advanced delivery systems are leading to new developments in pediatric vaccinology. Summarizes delivery systems currently in use and development, and prospects for the future. Broad overview of immunoengineering's impact on vaccinology, catering to Pediatric Clinicians and Immunologists.
Asunto(s)
Ingeniería Genética , Vacunación/métodos , Vacunas/administración & dosificación , Niño , Humanos , Vacunas/inmunologíaRESUMEN
RATIONALE: TRPM2 (transient receptor potential melastatin-2) expressed in endothelial cells (ECs) is a cation channel mediating Ca2+ entry in response to intracellular generation of adenosine diphosphoribose-the TRPM2 ligand. OBJECTIVE: Because polymorphonuclear neutrophils (PMN) interaction with ECs generates reactive oxygen species, we addressed the possible role of TRPM2 expressed in ECs in the mechanism of transendothelial migration of PMNs. METHODS AND RESULTS: We observed defective PMN transmigration in response to lipopolysaccharide challenge in adult mice in which the EC expressed TRPM2 is conditionally deleted (Trpm2iΔEC ). PMN interaction with ECs induced the entry of Ca2+ in ECs via the EC-expressed TRPM2. Prevention of generation of adenosine diphosphoribose in ECs significantly reduced Ca2+ entry in response to PMN activation of TRPM2 in ECs. PMNs isolated from gp91phox-/- mice significantly reduced Ca2+ entry in ECs via TRPM2 as compared with wild-type PMNs and failed to induce PMN transmigration. Overexpression of the adenosine diphosphoribose insensitive TRPM2 mutant channel (C1008âA) in ECs suppressed the Ca2+ entry response. Further, the forced expression of TRPM2 mutant channel (C1008âA) or silencing of poly ADP-ribose polymerase in ECs of mice prevented PMN transmigration. CONCLUSIONS: Thus, endotoxin-induced transmigration of PMNs was secondary to TRPM2-activated Ca2+ signaling and VE-cadherin phosphorylation resulting in the disassembly of adherens junctions and opening of the paracellular pathways. These results suggest blocking TRPM2 activation in ECs is a potentially important means of therapeutically modifying PMN-mediated vascular inflammation.
Asunto(s)
Células Endoteliales/metabolismo , Activación Neutrófila/fisiología , Neutrófilos/metabolismo , Canales Catiónicos TRPM/biosíntesis , Migración Transendotelial y Transepitelial/fisiología , Lesiones del Sistema Vascular/metabolismo , Animales , Movimiento Celular/fisiología , Células Cultivadas , Células Endoteliales/patología , Expresión Génica , Humanos , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Canales Catiónicos TRPM/genética , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patologíaRESUMEN
TNFα-stimulated gene-6 (TSG6), a 30-kDa protein generated by activated macrophages, modulates inflammation; however, its mechanism of action and role in the activation of macrophages are not fully understood. Here we observed markedly augmented LPS-induced inflammatory lung injury and mortality in TSG6-/- mice compared with WT (TSG6+/+) mice. Treatment of mice with intratracheal instillation of TSG6 prevented LPS-induced lung injury and neutrophil sequestration, and increased survival in mice. We found that TSG6 inhibited the association of TLR4 with MyD88, thereby suppressing NF-κB activation. TSG6 also prevented the expression of proinflammatory proteins (iNOS, IL-6, TNFα, IL-1ß, and CXCL1) while increasing the expression of anti-inflammatory proteins (CD206, Chi3l3, IL-4, and IL-10) in macrophages. This shift was associated with suppressed activation of proinflammatory transcription factors STAT1 and STAT3. In addition, we observed that LPS itself up-regulated the expression of TSG6 in TSG6+/+ mice, suggesting an autocrine role for TSG6 in transitioning macrophages. Thus, TSG6 functions by converting macrophages from a proinflammatory to an anti-inflammatory phenotype secondary to suppression of TLR4/NF-κB signaling and STAT1 and STAT3 activation.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Lesión Pulmonar/prevención & control , Macrófagos/inmunología , Animales , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Reprogramación Celular/inmunología , Inflamación/prevención & control , Mediadores de Inflamación/inmunología , Lipopolisacáridos/toxicidad , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/inmunología , Activación de Macrófagos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Fenotipo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismoRESUMEN
The influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, and endothelial cell biomechanics was investigated computationally and experimentally. The truncated tail phospholipids, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), are two major oxidation products of the unsaturated phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine. A combination of coarse-grained molecular dynamics simulations, Laurdan multiphoton imaging, and atomic force microscopy microindentation experiments was used to determine the impact of POVPC and PGPC on the structure of a multicomponent phospholipid bilayer and to assess the consequences of their incorporation on membrane packing and endothelial cell stiffness. Molecular simulations predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails, including decreased bilayer packing, decreased bilayer bending modulus, and increased water penetration. Disruption of lipid order was consistent with Laurdan imaging results indicating that POVPC and PGPC decrease the lipid packing of both ordered and disordered membrane domains. Computational predictions of a larger membrane perturbation effect by PGPC correspond to greater stiffness of PGPC-treated endothelial cells observed by measuring cellular elastic moduli using atomic force microscopy. Our results suggest that disruptions in membrane structure by oxidized phospholipids play a role in the regulation of overall endothelial cell stiffness.
Asunto(s)
Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Endoteliales/citología , Fenómenos Mecánicos/efectos de los fármacos , Éteres Fosfolípidos/farmacología , Animales , Fenómenos Biomecánicos/efectos de los fármacos , Bovinos , Membrana Celular/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Conformación Molecular , Simulación de Dinámica Molecular , Éteres Fosfolípidos/químicaRESUMEN
Vascular endothelial protein tyrosine phosphatase (VE-PTP) stabilizes endothelial adherens junctions (AJs) through constitutive dephosphorylation of VE-cadherin. Here we investigated the role of stromal interaction molecule 1 (STIM1) activation of store-operated Ca2+ entry (SOCE) in regulating AJ assembly. We observed that SOCE induced by STIM1 activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. Pyk2-induced tyrosine phosphorylation of VE-PTP promoted Src binding to VE-PTP, Src activation, and subsequent VE-cadherin phosphorylation and thereby increased the endothelial permeability response. The increase in permeability was secondary to disassembly of AJs. Pyk2-mediated responses were blocked in EC-restricted Stim1 knockout mice, indicating the requirement for STIM1 in initiating the signaling cascade. A peptide derived from the Pyk2 phosphorylation site on VE-PTP abolished the STIM1/SOCE-activated permeability response. Thus Pyk2 activation secondary to STIM1-induced SOCE causes tyrosine phosphorylation of VE-PTP, and VE-PTP, in turn, binds to and activates Src, thereby phosphorylating VE-cadherin to increase endothelial permeability through disassembly of AJs. Our results thus identify a novel signaling mechanism by which STIM1-induced Ca2+ signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin and hence increase endothelial permeability. Therefore, targeting the Pyk2 activation pathway may be a potentially important anti-inflammatory strategy.
Asunto(s)
Uniones Adherentes/metabolismo , Calcio/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar , Permeabilidad de la Membrana Celular , Células Endoteliales/metabolismo , Activación Enzimática , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Ratones Endogámicos C57BL , Microvasos/citología , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Péptidos/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Receptor PAR-1/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
A 24-year-old female patient presented to us with progressive dyspnea on exertion for last three year. She was not a known case of rheumatic heart disease. Her physical examination showed regular pulse and her blood pressure was 100/76 mm Hg. Cardiac palpation showed grade 3 parasternal heave and auscultation revelled an accentuated first heart sound, loud P2 and mid-diastolic long rumbling murmur at apex and pansystolic murmur of tricuspid regurgitation at lower left sterna border. Chest X-ray showed evidence of grade 3 pulmonary venous congestion. Transthoracic and transesophageal two-dimensional echocardiography revealed a double-orifice mitral valve of complete bridge type at the leaflet level. Both orifice sizes were unequal, with the anterolateral orifice being smaller than its counterpart. There was moderate subvalvular fusion and both commisures were fused. Color doppler examination showed two separate mitral diastolic flows with mean gradients of 22 mm and 20 mm of Hg, respectively. There was no mitral regurgitation and no left atrial or appendage clot was seen by transesophageal echocardiography. Transseptal puncture was done by the modified fluoroscopic method. Posteromedial orifice was crossed with a 24 mm Inoue balloon and dilated using the stepwise dilation technique. Anterolateral orifice was not crossed by Inuoe balloon after multiple attempts. A TYSHAK (NuMAD Canada Inc.) balloon (16 × 40mm) was taken over the wire and inflated successfully across the anterolateral orifice with the help of transthoracic echocardiography guidance. Mean gradient become 9 and 8 mm across the medial and lateral orifice. Patient was discharged in stable condition after two day. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Valvuloplastia con Balón , Cardiopatías Congénitas/complicaciones , Estenosis de la Válvula Mitral/terapia , Válvula Mitral/anomalías , Cardiopatía Reumática/terapia , Ecocardiografía Doppler en Color , Ecocardiografía Transesofágica , Femenino , Cardiopatías Congénitas/diagnóstico , Cardiopatías Congénitas/fisiopatología , Hemodinámica , Humanos , Válvula Mitral/diagnóstico por imagen , Válvula Mitral/fisiopatología , Estenosis de la Válvula Mitral/diagnóstico por imagen , Estenosis de la Válvula Mitral/etiología , Estenosis de la Válvula Mitral/fisiopatología , Cardiopatía Reumática/complicaciones , Cardiopatía Reumática/diagnóstico por imagen , Cardiopatía Reumática/fisiopatología , Resultado del Tratamiento , Adulto JovenRESUMEN
Stromal interacting molecule 1 (STIM1) regulates store-operated Ca(2+) entry (SOCE). Here we show that STIM1 expression in endothelial cells (ECs) is increased during sepsis and, therefore, contributes to hyperpermeability. LPS induced STIM1 mRNA and protein expression in human and mouse lung ECs. The induced STIM1 expression was associated with augmented SOCE as well as a permeability increase in both in vitro and in vivo models. Because activation of both the NF-κB and p38 MAPK signaling pathways downstream of TLR4 amplifies vascular inflammation, we studied the influence of these two pathways on LPS-induced STIM1 expression. Inhibition of either NF-κB or p38 MAPK activation by pharmacological agents prevented LPS-induced STIM1 expression. Silencing of the NF-κB proteins (p65/RelA or p50/NF-κB1) or the p38 MAPK isoform p38α prevented LPS-induced STIM1 expression and increased SOCE in ECs. In support of these findings, we found NF-κB and AP1 binding sites in the 5'-regulatory region of human and mouse STIM1 genes. Further, we demonstrated that LPS induced time-dependent binding of the transcription factors NF-κB (p65/RelA) and AP1 (c-Fos/c-Jun) to the STIM1 promoter. Interestingly, silencing of c-Fos, but not c-Jun, markedly reduced LPS-induced STIM1 expression in ECs. We also observed that silencing of p38α prevented c-Fos expression in response to LPS in ECs, suggesting that p38α signaling mediates the expression of c-Fos. These results support the proposal that cooperative signaling of both NF-κB and AP1 (via p38α) amplifies STIM1 expression in ECs and, thereby, contributes to the lung vascular hyperpermeability response during sepsis.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotoxinas/farmacología , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Animales , Secuencia de Bases , Permeabilidad Capilar , Células Cultivadas , Cartilla de ADN , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Molécula de Interacción Estromal 1 , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
A 22-year-old woman with severe mitral stenosis was referred to us for further evaluation and management. She was found to have severe mitral stenosis, severe tricuspid regurgitation with dilated right atrium and right ventricle with persistent left superior vena cava and hugely dilated coronary sinus. Valve was suitable for balloon mitral valvotomy. Cardiac catheterization showed interrupted inferior vena cava with azygos continuation to right atrium and large left superior vena cava draining to coronary sinus which was very much dilated. Right trans-jugular approach was tried for balloon mitral valvotomy, but was unsuccessful due to a very large right atrium and coronary sinus. Retrograde non trans-septal approach was used and balloon valvotomy was done successfully using a 24 mm × 40 mm TYSHAK balloon without any major complication. Reduction in the transmitral pressure gradient on cardiac catheterization data and transthoracic echocardiography confirmed successful procedure. Balloon mitral valvotomy can be done successfully in patients with the above unusual cardiac anatomy with no major procedural complications.
Asunto(s)
Valvuloplastia con Balón/métodos , Seno Coronario/patología , Estenosis de la Válvula Mitral/terapia , Malformaciones Vasculares/complicaciones , Vena Cava Inferior/anomalías , Vena Cava Superior/anomalías , Cateterismo Cardíaco , Seno Coronario/fisiopatología , Dilatación Patológica , Ecocardiografía Doppler en Color , Ecocardiografía Transesofágica , Femenino , Hemodinámica , Humanos , Estenosis de la Válvula Mitral/complicaciones , Estenosis de la Válvula Mitral/diagnóstico , Estenosis de la Válvula Mitral/fisiopatología , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Insuficiencia de la Válvula Tricúspide/complicaciones , Insuficiencia de la Válvula Tricúspide/diagnóstico , Malformaciones Vasculares/diagnóstico , Malformaciones Vasculares/fisiopatología , Vena Cava Inferior/fisiopatología , Vena Cava Superior/fisiopatología , Adulto JovenRESUMEN
Vaccination reduces morbidity and mortality due to infections, but efficacy may be limited due to distinct immunogenicity at the extremes of age. This raises the possibility of employing adjuvants to enhance immunogenicity and protection. Early IFNγ production is a hallmark of effective vaccine immunogenicity in adults serving as a biomarker that may predict effective adjuvanticity. We utilized mass cytometry (CyTOF) to dissect the source of adjuvant-induced cytokine production in human blood mononuclear cells (BMCs) from newborns (~39-week-gestation), adults (~18-63 years old) and elders (>65 years of age) after stimulation with pattern recognition receptors agonist (PRRa) adjuvants. Dimensionality reduction analysis of CyTOF data mapped the BMC compartment, elucidated age-specific immune responses and profiled PRR-mediated activation of monocytes and DCs upon adjuvant stimulation. Furthermore, we demonstrated PRRa adjuvants mediated innate IFNγ induction and mapped NK cells as the key source of TLR7/8 agonist (TLR7/8a) specific innate IFNγ responses. Hierarchical clustering analysis revealed age and TLR7/8a-specific accumulation of innate IFNγ producing γδ T cells. Our study demonstrates the application of mass cytometry and cutting-edge computational approaches to characterize immune responses across immunologically distinct age groups and may inform identification of the bespoke adjuvantation systems tailored to enhance immunity in distinct vulnerable populations.
Asunto(s)
Adyuvantes Inmunológicos , Leucocitos Mononucleares , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Adulto , Persona de Mediana Edad , Adyuvantes Inmunológicos/farmacología , Anciano , Adulto Joven , Adolescente , Interferón gamma/metabolismo , Recién Nacido , Femenino , Masculino , Factores de Edad , Inmunidad InnataRESUMEN
Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)-activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037's in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.
Asunto(s)
Adyuvantes Inmunológicos , Pirimidinas , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Humanos , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/metabolismo , Animales , Ratones , Adyuvantes Inmunológicos/farmacología , Receptor Toll-Like 7/agonistas , Pirimidinas/farmacología , Pirimidinas/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Imidazoles/farmacología , Imidazoles/química , Células THP-1 , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/inmunología , COVID-19/virología , COVID-19/inmunología , FN-kappa B/metabolismo , Femenino , Descubrimiento de Drogas/métodos , Inmunidad Innata/efectos de los fármacosRESUMEN
TGF-ß-activated kinase 1 (TAK1) plays crucial roles in innate and adaptive immune responses and is required for embryonic vascular development. However, TAK1's role in regulating vascular barrier integrity is not well defined. Here we show that endothelial TAK1 kinase function is required to maintain and repair the injured lung endothelial barrier. We observed that inhibition of TAK1 with 5Z-7-oxozeaenol markedly reduced expression of ß-catenin (ß-cat) and VE-cadherin at endothelial adherens junctions and augmented protease-activated receptor-1 (PAR-1)- or toll-like receptor-4 (TLR-4)-induced increases in lung vascular permeability. In inducible endothelial cell (EC)-restricted TAK1 knockout (TAK1i∆EC) mice, we observed that the lung endothelial barrier was compromised and in addition, TAK1i∆EC mice exhibited heightened sensitivity to septic shock. Consistent with these findings, we observed dramatically reduced ß-cat expression in lung ECs of TAK1i∆EC mice. Further, either inhibition or knockdown of TAK1 blocked PAR-1- or TLR-4-induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which in turn increased phosphorylation, ubiquitylation, and degradation of ß-cat in ECs to destabilize the endothelial barrier. Importantly, we showed that TAK1 inactivates GSK3ß through AKT activation in ECs. Thus our findings in this study point to the potential of targeting the TAK1-AKT-GSK3ß axis as a therapeutic approach to treat uncontrolled lung vascular leak during sepsis.
Asunto(s)
Receptor Toll-Like 4 , Lesiones del Sistema Vascular , Animales , Glucógeno Sintasa Quinasa 3 beta , Pulmón , Quinasas Quinasa Quinasa PAM , Ratones , Proteínas Proto-Oncogénicas c-aktRESUMEN
Motivation: Vaccines are a key biomedical intervention to prevent the spread of infectious diseases, but their efficacy can be limited by insufficient immunogenicity and nonuniform reactogenic profiles. Adjuvants are molecules that potentiate vaccine responses by inducing innate immune activation. However, there are a limited number of adjuvants in approved vaccines, and current approaches for preclinical adjuvant discovery and development are inefficient. To enhance adjuvant identification, we developed a protocol based on in vitro screening of human primary leukocytes. Summary: We describe a methodology utilizing high-throughput and high-content screening for novel adjuvant candidates that was used to screen a library of ~2,500 small molecules via a 384-well quantitative combined cytokine and flow cytometry immunoassay in primary human peripheral blood mononuclear cells (PBMCs) from 4 healthy adult study participants. Hits were identified based on their induction of soluble cytokine (TNF, IFNg and IL-10) secretion and PBMC maturation (CD 80/86, Ox40, and HLA-DR) in at least two of the four donors screened. From an initial set of 197 compounds identified using these biomarkers-an 8.6% hit rate-we downselected to five scaffolds that demonstrated robust efficacy and potency in vitro and evaluated the top hit, vinblastine sulfate, for adjuvanticity in vivo. Vinblastine sulfate significantly enhanced murine humoral responses to recombinant SARS-CoV-2 spike protein, including a four-fold enhancement of IgG titer production when compared to treatment with the spike antigen alone. Overall, we outline a methodology for discovering immunomodulators with adjuvant potential via high-throughput screening of PBMCs in vitro that yielded a lead compound with in vivo adjuvanticity.
RESUMEN
Infection is the most common cause of mortality early in life, yet the broad potential of immunization is not fully realized in this vulnerable population. Most vaccines are administered during infancy and childhood, but in some cases the full benefit of vaccination is not realized in-part. New adjuvants are cardinal to further optimize current immunization approaches for early life. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) agonist adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is urgently required because of the resurgence of pertussis in many countries, contemporaneous to the switch from whole cell to acellular vaccines. In this context, TLR7/8 adjuvant based vaccine formulation strategies may be a promising tool to enhance and accelerate early life immunity by acellular B. pertussis vaccines. In the present study, we optimized (a) the formulation delivery system, (b) structure, and (c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes, including dendritic cells. Upon immunization of neonatal mice, this TLR7/8 adjuvant overcame neonatal hyporesponsiveness to acellular pertussis vaccination by driving a T helper (Th)1/Th17 biased T cell- and IgG2c-skewed humoral response to a licensed acellular vaccine (DTaP). This potent immunization strategy may represent a new paradigm for effective immunization against pertussis and other pathogens in early life.
Asunto(s)
Tos Ferina , Animales , Niño , Humanos , Lactante , Ratones , Adyuvantes Inmunológicos/farmacología , Adyuvantes Farmacéuticos , Vacuna contra la Tos Ferina , Receptor Toll-Like 7/agonistas , Vacunación , Vacunas Acelulares , Tos Ferina/epidemiologíaRESUMEN
Adjuvanted nanocarrier-based vaccines hold substantial potential for applications in novel early-life immunization strategies. Here, via mouse and human age-specific in vitro modeling, we identified the combination of a small-molecule STING agonist (2'3'-cyclic GMP-AMP, cGAMP) and a TLR7/8 agonist (CL075) to drive the synergistic activation of neonatal dendritic cells and precision CD4 T-helper (Th) cell expansion via the IL-12/IFNγ axis. We further demonstrate that the vaccination of neonatal mice with quadrivalent influenza recombinant hemagglutinin (rHA) and an admixture of two polymersome (PS) nanocarriers separately encapsulating cGAMP (cGAMP-PS) and CL075 (CL075-PS) drove robust Th1 bias, high frequency of T follicular helper (TFH) cells, and germinal center (GC) B cells along with the IgG2c-skewed humoral response in vivo. Dual-loaded cGAMP/CL075-PSs did not outperform admixed cGAMP-PS and CL075-PS in vivo. These data validate an optimally designed adjuvantation system via age-selected small-molecule synergy and a multicomponent nanocarrier formulation as an effective approach to induce type 1 immune responses in early life.
Asunto(s)
Hemaglutininas , Receptor Toll-Like 7 , Adyuvantes Inmunológicos/farmacología , Animales , Humanos , Inmunización , Interleucina-12 , Ratones , VacunaciónRESUMEN
mRNA vaccines have been key to addressing the SARS-CoV-2 pandemic but have impaired immunogenicity and durability in vulnerable older populations. We evaluated the mRNA vaccine BNT162b2 in human in vitro whole blood assays with supernatants from adult (18-50 years) and elder (≥60 years) participants measured by mass spectrometry and proximity extension assay proteomics. BNT162b2 induced increased expression of soluble proteins in adult blood (e.g., C1S, PSMC6, CPN1), but demonstrated reduced proteins in elder blood (e.g., TPM4, APOF, APOC2, CPN1, and PI16), including 30-85% lower induction of TH1-polarizing cytokines and chemokines (e.g., IFNγ, and CXCL10). Elder TH1 impairment was validated in mice in vivo and associated with impaired humoral and cellular immunogenicity. Our study demonstrates the utility of a human in vitro platform to model age-specific mRNA vaccine activity, highlights impaired TH1 immunogenicity in older adults, and provides rationale for developing enhanced mRNA vaccines with greater immunogenicity in vulnerable populations.
RESUMEN
Traditional vaccine development against infectious diseases has been guided by the overarching aim to generate efficacious vaccines normally indicated by an antibody and/or cellular response that correlates with protection. However, this approach has been shown to be only a partially effective measure, since vaccine- and pathogen-specific immunity may not perfectly overlap. Thus, some vaccine development strategies, normally focused on targeted generation of both antigen specific antibody and T cell responses, resulting in a long-lived heterogenous and stable pool of memory lymphocytes, may benefit from better mimicking the immune response of a natural infection. However, challenges to achieving this goal remain unattended, due to gaps in our understanding of human immunity and full elucidation of infectious pathogenesis. In this review, we describe recent advances in the development of effective vaccines, focusing on how understanding the differences in the immunizing and non-immunizing immune responses to natural infections and corresponding shifts in immune ontogeny are crucial to inform the next generation of infectious disease vaccines.
Asunto(s)
Señales (Psicología) , Vacunas , Humanos , Inmunidad Innata , Linfocitos TRESUMEN
Other than clean drinking water, vaccines have been the most effective public health intervention in human history, yet their full potential is still untapped. To date, vaccine development has been largely limited to empirical approaches focused on infectious diseases and has targeted entire populations, potentially disregarding distinct immunity in vulnerable populations such as infants, elders, and the immunocompromised. Over the past few decades innovations in genetic engineering, adjuvant discovery, formulation science, and systems biology have fueled rapid advances in vaccine research poised to consider demographic factors (e.g., age, sex, genetics, and epigenetics) in vaccine discovery and development. Current efforts are focused on leveraging novel approaches to vaccine discovery and development to optimize vaccinal antigen and, as needed, adjuvant systems to enhance vaccine immunogenicity while maintaining safety. These approaches are ushering in an era of precision vaccinology aimed at tailoring immunization for vulnerable populations with distinct immunity. To foster collaboration among leading vaccinologists, government, policy makers, industry partners, and funders from around the world, the Precision Vaccines Program at Boston Children's Hospital hosted the 2nd International Precision Vaccines Conference (IPVC) at Harvard Medical School on the 17th-18th October 2019. The conference convened experts in vaccinology, including vaccine formulation and adjuvantation, immunology, cell signaling, systems biology, biostatistics, bioinformatics, as well as vaccines for non-infectious indications such as cancer and opioid use disorder. Herein we review highlights from the 2nd IPVC and discuss key concepts in the field of precision vaccines.
Asunto(s)
Medicina de Precisión , Vacunas , Animales , HumanosRESUMEN
Infections are most frequent at the extremes of life, especially among newborns, reflecting age-specific differences in immunity. Monocytes maintain tissue-homeostasis and defence-readiness by escaping circulation in the absence of inflammation to become tissue-resident antigen presenting cells in vivo. Despite equivalent circulating levels, neonates demonstrate lower presence of monocytes inside peripheral tissues as compared to adults. To study the ability of monocytes to undergo autonomous transendothelial extravasation under biologically accurate circumstances we engineered a three-dimensional human vascular-interstitial model including collagen, fibronectin, primary endothelial cells and autologous untreated plasma. This microphysiological tissue construct enabled age-specific autonomous extravasation of monocytes through a confluent human endothelium in the absence of exogenous chemokines and activation. Both CD16- and CD16+ newborn monocytes demonstrated lower adherence and extravasation as compared to adults. In contrast, pre-activated tissue constructs were colonized by newborn monocytes at the same frequency than adult monocytes, suggesting that neonatal monocytes are capable of colonizing inflamed tissues. The presence of autologous plasma neither improved newborn homeostatic extravasation nor shaped age-specific differences in endothelial cytokines that could account for this impairment. Newborn monocytes demonstrated significantly lower surface expression of CD31 and CD11b, and mechanistic experiments using blocking antibodies confirmed a functional role for CD31 and CD54 in neonatal homeostatic extravasation. Our data suggests that newborn monocytes are intrinsically impaired in extravasation through quiescent endothelia, a phenomenon that could contribute to the divergent immune responsiveness to vaccines and susceptibility to infection observed during early life.