RESUMEN
Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.
Asunto(s)
Aciltransferasas/inmunología , Evasión Inmune/inmunología , Inmunidad Innata/inmunología , Lípido A/inmunología , Yersinia pestis/inmunología , Animales , Evolución Biológica , Línea Celular , Línea Celular Tumoral , Células HEK293 , Humanos , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Polimorfismo de Nucleótido Simple/inmunología , Células THP-1/inmunología , Células U937 , Yersinia pseudotuberculosis/inmunologíaRESUMEN
Immune sensing of the Gram-negative bacterial membrane glycolipid lipopolysaccharide (LPS) is both a critical component of host defense against bacterial infection and a contributor to the hyperinflammatory response, potentially leading to sepsis and death. Innate immune activation by LPS is due to the lipid A moiety, an acylated di-glucosamine molecule that can activate inflammatory responses via the extracellular sensor Toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) or the cytosolic sensor caspase-11 (Casp11). The number and length of acyl chains present on bacterial lipid A structures vary across bacterial species and strains, which affects the magnitude of TLR4 and Casp11 activation. TLR4 and Casp11 are thought to respond similarly to various lipid A structures, as tetra-acylated lipid A structures do not activate either sensor, whereas hexa-acylated structures activate both sensors. However, the precise features of lipid A that determine the differential activation of each receptor remain poorly defined, as direct analysis of extracellular and cytosolic responses to the same sources and preparations of LPS/lipid A structures have been limited. To address this question, we used rationally engineered lipid A isolated from a series of bacterial acyl-transferase mutants that produce novel, structurally defined molecules. Intriguingly, we found that the location of specific secondary acyl chains on lipid A resulted in differential recognition by TLR4 or Casp11, providing new insight into the structural features of lipid A required to activate either TLR4 or Casp11. Our findings indicate that TLR4 and Casp11 sense nonoverlapping areas of lipid A chemical space, thereby constraining the ability of Gram-negative pathogens to evade innate immunity.
Asunto(s)
Lípido A , Receptor Toll-Like 4 , Acilación , Animales , Caspasas , Lípido A/química , Lipopolisacáridos , Ratones , Receptor Toll-Like 4/metabolismoRESUMEN
Detection of Gram-negative bacterial lipid A by the extracellular sensor, myeloid differentiation 2 (MD2)/Toll-like receptor 4 (TLR4), or the intracellular inflammasome sensors, CASP4 and CASP5, induces robust inflammatory responses. The chemical structure of lipid A, specifically its phosphorylation and acylation state, varies across and within bacterial species, potentially allowing pathogens to evade or suppress host immunity. Currently, it is not clear how distinct alterations in the phosphorylation or acylation state of lipid A affect both human TLR4 and CASP4/5 activation. Using a panel of engineered lipooligosaccharides (LOS) derived from Yersinia pestis with defined lipid A structures that vary in their acylation or phosphorylation state, we identified that differences in phosphorylation state did not affect TLR4 or CASP4/5 activation. However, the acylation state differentially impacted TLR4 and CASP4/5 activation. Specifically, all tetra-, penta-, and hexa-acylated LOS variants examined activated CASP4/5-dependent responses, whereas TLR4 responded to penta- and hexa-acylated LOS but did not respond to tetra-acylated LOS or penta-acylated LOS lacking the secondary acyl chain at the 3' position. As expected, lipid A alone was sufficient for TLR4 activation. In contrast, both core oligosaccharide and lipid A were required for robust CASP4/5 inflammasome activation in human macrophages, whereas core oligosaccharide was not required to activate mouse macrophages expressing CASP4. Our findings show that human TLR4 and CASP4/5 detect both shared and nonoverlapping LOS/lipid A structures, which enables the innate immune system to recognize a wider range of bacterial LOS/lipid A and would thereby be expected to constrain the ability of pathogens to evade innate immune detection.
Asunto(s)
Lípido A , Receptor Toll-Like 4 , Acilación , Animales , Humanos , Inflamasomas , Lípido A/química , Lipopolisacáridos , Macrófagos , Ratones , Receptor Toll-Like 4/metabolismoRESUMEN
Sepsis remains one of the most lethal and costly conditions treated in U.S. hospitals, with approximately 50% of cases caused by Gram-negative bacterial infections. Septic shock is induced when lipopolysaccharide (LPS), the main component of Gram-negative outer bacterial membrane, signals through the Toll-like receptor 4 (TLR4) complex. Lethal endotoxemia, a model for septic shock, was induced in WT C57BL6 and TLR4-/- mice by administration of Escherichia coli LPS. WT LPS treated mice showed high morbidity, while PBS treated LPS and treated TLR4-/- mice did not. ANOVA analysis of label-free quantification of longitudinal serum proteome revealed 182 out of 324 proteins in LPS injected WT mice that were significantly changed across four time points (0, 6, 12, and 18 h). No significant changes were identified in the two control groups. From the 182 identified proteins, examples of known sepsis biomarkers were validated by ELISA, which showed similar trends as MS proteomics data. Longitudinal analysis within individual mice produced 3-fold more significantly changed proteins than pair-wise comparison. A subsequent global analysis of WT and TLR4-/- mice identified pathways activated independent of TLR4. These pathways represent possible compensatory mechanisms that allow for control of Gram-negative bacterial infection regardless of host immune status.
Asunto(s)
Sepsis , Choque Séptico , Animales , Lipopolisacáridos/toxicidad , Ratones , Proteómica , Sepsis/genética , Receptor Toll-Like 4/genéticaRESUMEN
Infectious diseases propagated by arthropod vectors, such as tularemia, are commonly initiated via dermal infection of the skin. However, due to the technical difficulties in achieving accurate and reproducible dermal deposition, intradermal models are less commonly used. To overcome these limitations, we used microneedle arrays (MNAs), which are micron-scale polymeric structures, to temporarily disrupt the barrier function of the skin and deliver a bacterial inoculum directly to the dermis of an animal. MNAs increase reliability by eliminating leakage of the inoculum or blood from the injection site, thereby providing a biologically relevant model for arthropod-initiated disease. Here, we validate the use of MNAs as a means to induce intradermal infection using a murine model of tularemia initiated by Francisella novicida We demonstrate targeted delivery of the MNA bolus to the dermal layer of the skin, which subsequently led to innate immune cell infiltration. Additionally, F. novicida-coated MNAs were used to achieve lethality in a dose-dependent manner in C57BL/6 mice. The immune profile of infected mice mirrored that of established F. novicida infection models, consisting of markedly increased serum levels of interleukin-6 and keratinocyte chemoattractant, splenic T-cell depletion, and an increase in splenic granulocytes, together confirming that MNAs can be used to reproducibly induce tularemia-like pathogenesis in mice. When MNAs were used to immunize mice using an attenuated F. novicida mutant (F. novicida ΔlpxD1), all immunized mice survived a lethal subcutaneous challenge. Thus, MNAs can be used to effectively deliver viable bacteria in vivo and provide a novel avenue to study intradermally induced microbial diseases in animal models.
Asunto(s)
Francisella/patogenicidad , Inyecciones Intradérmicas/instrumentación , Agujas , Piel/microbiología , Tularemia/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Dermis/inmunología , Dermis/microbiología , Modelos Animales de Enfermedad , Femenino , Inmunización/instrumentación , Inmunización/métodos , Interleucina-6/sangre , Ratones , Ratones Endogámicos C57BL , Mutación , Reproducibilidad de los Resultados , Piel/inmunología , Bazo/inmunología , Tularemia/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
UV radiation (UVR) induces DNA damage, leading to the accumulation of mutations in epidermal keratinocytes and immunosuppression, which contribute to the development of nonmelanoma skin cancer. We reported previously that the TLR4-MyD88 signaling axis is necessary for UV-induced apoptosis. In the dinitrofluorobenzene contact hypersensitivity model, UV-irradiated MyD88-deficient (MyD88(-/-)) C57BL/6 mice had intact ear swelling, exaggerated inflammation, and higher levels of dinitrofluorobenzene-specific IgG2a compared with wild-type (WT) mice. Even with normal UV-induced, dendritic cell migration, DNA damage in the local lymph nodes was less pronounced in MyD88(-/-) mice compared with WT mice. Cultured, UV-irradiated WT APCs showed cleavage (inactivation) of the DNA damage-recognition molecule PARP, whereas PARP persisted in MyD88(-/-) and TLR4(-/-) APCs. Epidermal DNA from in vivo UV-irradiated MyD88(-/-) mice had an increased resolution rate of cyclobutane pyrimidine dimers. Both in vitro treatment of MyD88(-/-) APCs with and intradermal in vivo injections of PARP inhibitor, PJ-34, caused WT-level cyclobutane pyrimidine dimer repair. Lymphoblasts deficient in DNA repair (derived from a xeroderma pigmentosum group A patient) failed to augment DNA repair after MyD88 knockdown after UVR, in contrast to lymphoblasts from a healthy control. These data suggest that interference with the TLR4/MyD88 pathway may be a useful tool in promoting DNA repair and maintaining immune responses following UVR-induced damage.
Asunto(s)
Reparación del ADN , Terapia de Inmunosupresión , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/efectos de la radiación , Piel/inmunología , Piel/metabolismo , Receptor Toll-Like 4/metabolismo , Rayos Ultravioleta , Animales , Microambiente Celular/genética , Microambiente Celular/inmunología , Microambiente Celular/efectos de la radiación , Daño del ADN/efectos de la radiación , Femenino , Humanos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/efectos de la radiación , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Piel/efectos de la radiación , Receptor Toll-Like 4/genéticaRESUMEN
UVR-induced apoptosis in cutaneous antigen presenting cells (APC) causes systemic immune suppression and is dependent on TLR4/MyD88 signalling, but the apoptotic signalling pathways have not been defined. Macrophages pretreated with lipopolysaccharide (LPS) were unresponsive to subsequent LPS treatment, however, but were susceptible to UVR-induced apoptosis. Macrophage survival and apoptotic events after UVR were also unaffected by treatment with TLR4 antagonists, a blocking IgG or a TLR4 analog antagonist, suggesting that UVR cell death is independent of a soluble ligand. After UVR, IRAK4KDKI (catalytically inactive IRAK4) and wild-type (WT) macrophages show equivalent levels of survival, as measured by MTT assay, and apoptosis, as measured by cleaved caspase-3. Furthermore, in macrophages from both mice, UVR activated caspase-8 and PARP, while inactivating Rip3. This finding is supported by a lack of IRAK1 degradation after UVR, compared to treatment with TLR2 or TLR4 agonists. UVR induced association of MyD88 with FADD, an extrinsic apoptotic pathway protein, but not IRAK4. UVR-induced migration of FADD to the cell membrane of WT macrophages, but not MyD88-/- macrophages, was observed (confocal microscopy). Co-immunoprecipitation using an epitope-tagged MyD88 revealed that FADD, but not TRADD, was recruited to MyD88 within 30 minutes of UVR exposure. UVR engages TLR4/MyD88 as a death signalling complex, rather than the classical inflammatory signalling pathway triggered by PAMP recognition of TLR4. These studies provide the rationale for the future development of topical TLR4 modulating therapies to interfere with this UVB-mediated apoptosis and the associated negative consequences of immune suppression.
Asunto(s)
Apoptosis , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Macrófagos Peritoneales/efectos de la radiación , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Femenino , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Lipopolisacáridos , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BLRESUMEN
Human Herpesvirus 6 (HHV-6) is a ubiquitous virus with an estimated seroprevalence of 95% in the adult population. HHV-6 is associated with several neurologic disorders, including multiple sclerosis, an inflammatory demyelinating disease affecting the CNS. Animal models of HHV-6 infection would help clarify its role in human disease but have been slow to develop because rodents lack CD46, the receptor for cellular entry. Therefore, we investigated the effects of HHV-6 infections in a non-human primate, the common marmoset Callithrix jacchus. We inoculated a total of 12 marmosets with HHV-6A and HHV-6B intravenously and HHV-6A intranasally. Animals were monitored for 25 weeks post-inoculation clinically, immunologically and by MRI. Marmosets inoculated with HHV-6A intravenously exhibited neurologic symptoms and generated virus-specific antibody responses, while those inoculated intravenously with HHV-6B were asymptomatic and generated comparatively lower antibody responses. Viral DNA was detected at a low frequency in paraffin-embedded CNS tissue of a subset of marmosets inoculated with HHV-6A and HHV-6B intravenously. When different routes of HHV-6A inoculation were compared, intravenous inoculation resulted in virus-specific antibody responses and infrequent detection of viral DNA in the periphery, while intranasal inoculation resulted in negligible virus-specific antibody responses and frequent detection of viral DNA in the periphery. Moreover, marmosets inoculated with HHV-6A intravenously exhibited neurologic symptoms, while marmosets inoculated with HHV-6A intranasally were asymptomatic. We demonstrate that a marmoset model of HHV-6 infection can serve to further define the contribution of this ubiquitous virus to human neurologic disorders.
Asunto(s)
Callithrix/fisiología , Modelos Animales de Enfermedad , Herpesvirus Humano 6/patogenicidad , Enfermedades del Sistema Nervioso/inmunología , Infecciones por Roseolovirus , Administración Intranasal , Animales , Encéfalo/patología , Encéfalo/virología , ADN Viral/análisis , Herpesvirus Humano 6/fisiología , Humanos , Inyecciones Intravenosas , Imagen por Resonancia Magnética , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/virología , Infecciones por Roseolovirus/diagnóstico , Infecciones por Roseolovirus/inmunología , Infecciones por Roseolovirus/virología , Médula Espinal/patología , Médula Espinal/virologíaRESUMEN
Viruses have been implicated in the development of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and multiple sclerosis. Human herpesvirus-6 (HHV-6) is a neurotropic virus that has been associated with a wide variety of neurologic disorders, including encephalitis, mesial temporal lobe epilepsy, and multiple sclerosis. Currently, the route of HHV-6 entry into the CNS is unknown. Using autopsy specimens, we found that the frequency of HHV-6 DNA in the olfactory bulb/tract region was among the highest in the brain regions examined. Given this finding, we investigated whether HHV-6 may infect the CNS via the olfactory pathway. HHV-6 DNA was detected in a total of 52 of 126 (41.3%) nasal mucous samples, showing the nasal cavity is a reservoir for HHV-6. Furthermore, specialized olfactory-ensheathing glial cells located in the nasal cavity were demonstrated to support HHV-6 replication in vitro. Collectively, these results support HHV-6 utilization of the olfactory pathway as a route of entry into the CNS.
Asunto(s)
Sistema Nervioso Central/virología , ADN Viral/análisis , Herpesvirus Humano 6/fisiología , Vías Olfatorias/virología , Internalización del Virus , Encéfalo/virología , Humanos , Membrana Mucosa/virología , Neuroglía/virología , Replicación ViralRESUMEN
Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in severe morbidity and mortality. We have previously shown that the Bacterial Enzymatic Combinatorial Chemistry (BECC) adjuvants, BECC438 and BECC470, formulated with an influenza virus hemagglutinin (HA) protein vaccine, offer greater protection from influenza virus challenge in mouse respiratory models using adult mice than standard HA:adjuvant combinations. In this study, we determined that immunization with HA + BECC adjuvants also significantly broadened the epitopes targeted on HA as compared with other adjuvants, resulting in increased titers of antibodies directed against the highly conserved HA stalk domain. Importantly, we demonstrate that BECC470 combined with an influenza virus HA protein antigen in a prime-only immunization regimen was able to achieve complete protection from challenge in a ~ 12-month-old mouse aged model. Together, this demonstrates the heightened protection provided by the BECC470 adjuvant in an influenza virus vaccine model and shows the enhanced immune response, as compared to other adjuvants elicited by the formulation of HA with BECC470.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae , Animales , Humanos , Ratones , Adyuvantes Inmunológicos , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas , Gripe Humana , Ratones Endogámicos BALB C , Receptor Toll-Like 4RESUMEN
Conventional CD8+ T-lymphocytes are thought to be major effector cells in allergic contact dermatitis (ACD). However, previous work has demonstrated a significant population of invariant natural killer T-cells (iNKT-cells) in the elicitation phase of ACD. In this study, we investigate whether iNKT-cells have the capacity to serve as effector lymphocytes in ACD. Using in situ staining of skin biopsy specimens from ACD lesions, we observed intra-epidermal iNKT-cells. Presence of these cells provides the possibility of interactions with keratinocytes (KC), Langerhans cells (LC) and CD1d-bearing antigen-presenting cells (APC). Investigation into gene expression profiles of cytotoxic effector molecules in seven different cases of ACD found that the expression of perforin and granzymes A, B and K were significantly elevated in ACD relative to paired clinically normal skin. Immunostaining of ACD skin biopsy specimens revealed that these cytotoxic granules indeed localized to iNKT-cells. Studies of antigen presentation of KC to iNKT-cells show that these epithelial cells do not activate the expression of cytotoxicity effector genes in resting iNKT-cells, but had the capacity to serve as targets for activated iNKT-cells, which was dependent on CD1d expression. Mature LC were not able to present glycolipids to iNKT-cells and did not up-regulate CD1d in vitro to a variety of maturational stimuli or in vivo during ACD. These data suggest that iNKT-cells can serve as effector cells during human ACD and provide the rationale for developing inhibitory glycolipids as therapeutic agents for ACD.
Asunto(s)
Antígenos CD1d/inmunología , Comunicación Celular/inmunología , Dermatitis Alérgica por Contacto/inmunología , Queratinocitos/inmunología , Células T Asesinas Naturales/inmunología , Adulto , Anciano , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Biopsia , Células Cultivadas , Gránulos Citoplasmáticos/inmunología , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/patología , Dermatitis Alérgica por Contacto/metabolismo , Dermatitis Alérgica por Contacto/patología , Epidermis/inmunología , Epidermis/metabolismo , Epidermis/patología , Femenino , Glucolípidos/inmunología , Glucolípidos/metabolismo , Humanos , Queratinocitos/patología , Células de Langerhans/inmunología , Células de Langerhans/patología , Masculino , Persona de Mediana Edad , Células T Asesinas Naturales/patología , Péptido Hidrolasas/inmunología , Péptido Hidrolasas/metabolismo , Perforina/inmunología , Perforina/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in hospitalization or death. In this study, TLR4 immunostimulatory molecules, Bacterial Enzymatic Combinatorial Chemistry (BECC) 438 and BECC470 were found to be superior IAV vaccine adjuvants when compared to the classic adjuvant alhydrogel (alum) and Phosphorylated Hexa-Acyl Disaccharide (PHAD), a synthetic TLR4 agonist. BECC molecules allow for antigen sparing of a recombinant HA (rHA) protein, elicit a more balanced IgG1/IgG2a response, and were protective in a prime only dosing schedule. Importantly, BECC molecules afford protection from a heterologous IAV strain demonstrating that a cross-protective influenza vaccine is possible when the antigen is effectively adjuvanted.
Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Infecciones por Orthomyxoviridae , Adyuvantes Inmunológicos , Anticuerpos Antivirales , Humanos , Inmunoglobulina G , Gripe Humana/prevención & control , Infecciones por Orthomyxoviridae/prevención & control , Receptor Toll-Like 4RESUMEN
Current human papilloma virus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain that lack vaccine coverage. The novel RG1-VLP (virus-like particle) vaccine candidate utilizes the HPV16-L1 subunit as a backbone to display an inserted HPV16-L2 17-36 a.a. "RG1" epitope; the L2 RG1 epitope is conserved across many HPV types and the generation of cross-neutralizing antibodies (Abs) against which has been demonstrated. In an effort to heighten the immunogenicity of the RG1-VLP vaccine, we compared in BALB/c mice adjuvant formulations consisting of novel bacterial enzymatic combinatorial chemistry (BECC)-derived toll-like receptor 4 (TLR4) agonists and the aluminum hydroxide adjuvant Alhydrogel. In the presence of BECC molecules, consistent improvements in the magnitude of Ab responses to both HPV16-L1 and the L2 RG1 epitope were observed compared to Alhydrogel alone. Furthermore, neutralizing titers to HPV16 as well as cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39 were augmented in the presence of BECC agonists as well. Levels of L1 and L2-specific Abs were achieved after two vaccinations with BECC/Alhydrogel adjuvant that were equivalent to or greater than levels achieved with 3 vaccinations with Alhydrogel alone, indicating that the presence of BECC molecules resulted in accelerated immune responses that could allow for a decreased dose schedule for VLP-based HPV vaccines. In addition, dose-sparing studies indicated that adjuvantation with BECC/Alhydrogel allowed for a 75% reduction in antigen dose while still retaining equivalent magnitudes of responses to the full VLP dose with Alhydrogel. These data suggest that adjuvant optimization of HPV VLP-based vaccines can lead to rapid immunity requiring fewer boosts, dose-sparing of VLPs expensive to produce, and the establishment of a longer-lasting humoral immunity.
Asunto(s)
Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Vacunas contra Papillomavirus , Vacunas de Partículas Similares a Virus , Animales , Anticuerpos Antivirales , Proteínas de la Cápside , Ratones , Ratones Endogámicos BALB C , Papillomaviridae , Infecciones por Papillomavirus/prevención & control , Receptor Toll-Like 4RESUMEN
Toll-like receptors (TLRs), a family of "pattern recognition receptors," bind microbial and host-derived molecules, leading to intracellular signaling and proinflammatory gene expression. TLR4 is unique in that ligand-mediated activation requires the co-receptor myeloid differentiation 2 (MD2) to initiate two signaling cascades: the MyD88-dependent pathway is initiated at the cell membrane, and elicits rapid MAP kinase and NF-κB activation, while the TIR-domain containing adaptor inducing interferon-ß (TRIF)-dependent pathway is initiated from TLR4-containing endosomes and results in IRF3 activation. Previous studies associated inflammation with the MyD88 pathway and adjuvanticity with the TRIF pathway. Gram-negative lipopolysaccharide (LPS) is a potent TLR4 agonist, and structurally related molecules signal through TLR4 to differing extents. Herein, we compared monophosphoryl lipid A (sMPL) and E6020, two synthetic, non-toxic LPS lipid A analogs used as vaccine adjuvants, for their capacities to activate TLR4-mediated innate immune responses and to enhance antibody production. In mouse macrophages, high dose sMPL activates MyD88-dependent signaling equivalently to E6020, while E6020 exhibits significantly more activation of the TRIF pathway (a "TRIF bias") than sMPL. Eritoran, a TLR4/MD2 antagonist, competitively inhibited sMPL more strongly than E6020. Despite these differences, sMPL and E6020 adjuvants enhanced antibody responses to comparable extents, with balanced immunoglobulin (Ig) isotypes in two immunization models. These data indicate that a TRIF bias is not necessarily predictive of superior adjuvanticity.
Asunto(s)
Factor 88 de Diferenciación Mieloide , Receptor Toll-Like 4 , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Trastornos Disociativos , Lipopolisacáridos , Ratones , Receptor Toll-Like 4/metabolismo , Receptores Toll-LikeRESUMEN
In the preceding article "Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate" by Oyler et al., an error in the J5 E. coli LPS chemical structure (Figs. 2 and 4) was introduced and propagated into the final revision.
RESUMEN
Vaccination can significantly reduce worldwide morbidity and mortality to infectious diseases, thereby reducing the health burden as a result of microbial infections. Effective vaccines contain three components: a delivery system, an antigenic component of the pathogen, and an adjuvant. With the growing use of purely recombinant or synthetic antigens, there is a need to develop novel adjuvants that enhance the protective efficacy of a vaccine against infection. Using a structure-activity relationship (SAR) model, we describe here the synthesis of a novel TLR4 ligand adjuvant compound, BECC438, by bacterial enzymatic combinatorial chemistry (BECC). This compound was identified using an in vitro screening pipeline consisting of (i) NFκB activation and cytokine production by immortalized cell lines, (ii) cytokine production by primary human PBMCs, and (iii) upregulation of surface costimulatory markers by primary human monocyte-derived dendritic cells. Using this SAR screening regimen, BECC438 was shown to produce an innate immune activation profile comparable to the well-characterized TLR4 agonist adjuvant compound, phosphorylated hexa-acyl disaccharide (PHAD). To evaluate the in vivo adjuvant activity of BECC438, we used the known protective Yersinia pestis (Yp) antigen, rF1-V, in a murine prime-boost vaccination schedule followed by lethal challenge. In addition to providing protection from lethal challenge, BECC438 stimulated production of higher levels of rF1-V-specific total IgG as compared to PHAD after both prime and boost vaccinations. Similar to PHAD, BECC438 elicited a balanced IgG1/IgG2c response, indicative of active TH2/TH1-driven immunity. These data demonstrate that the novel BECC-derived TLR4L adjuvant, BECC438, elicits cytokine profiles in vitro similar to PHAD, induces high antigen-specific immune titers and a TH1-associated IgG2c immune titer skew, and protects mice against a lethal Yp challenge.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Lípido A/química , Vacuna contra la Peste/inmunología , Peste/prevención & control , Receptor Toll-Like 4/agonistas , Adyuvantes Inmunológicos/química , Animales , Anticuerpos Antibacterianos/sangre , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inmunoglobulina G/sangre , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Ratones Endogámicos C57BL , Vacuna contra la Peste/administración & dosificación , Relación Estructura-Actividad , Análisis de Supervivencia , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS3 analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts. Graphical Abstract á .
Asunto(s)
Vacunas contra Escherichia coli/química , Escherichia coli/química , Lipopolisacáridos/química , Espectrometría de Masas en Tándem/métodos , Línea Celular , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Humanos , Lipopolisacáridos/inmunologíaRESUMEN
Adjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A) are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential sources among bacterial strains, the number of useable compounds within this promising class of adjuvants are few. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate rationally designed, functionally diverse lipid A. BECC removes endogenous or introduces exogenous lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis to develop structurally distinct LOS molecules that elicit differential Toll-like receptor 4 (TLR4) activation. Using reporter cell lines that measure NF-κB activation, BECC-derived molecules were screened for the ability to induce a lower proinflammatory response than Escherichia coli LOS. Their structures exhibit varied, dose-dependent, TLR4-driven NF-κB activation with both human and mouse TLR4 complexes. Additional cytokine secretion screening identified molecules that induce levels of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) comparable to the levels induced by phosphorylated hexa-acyl disaccharide (PHAD). The lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary blood mononuclear cells (PBMCs), and human monocyte-derived dendritic cells (DCs). This newly described system allows directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates.IMPORTANCE There is an urgent need to develop effective vaccines against infectious diseases that continue to be major causes of morbidity and mortality worldwide. Making effective vaccines requires selecting an adjuvant to strengthen an appropriate and protective immune response. This work describes a practical method, bacterial enzymatic combinatorial chemistry (BECC), for generating functionally diverse molecules for adjuvant use. These molecules were analyzed in cell culture for their ability to initiate immune stimulatory activity. Several of the assays described herein show promising in vitro cytokine production and costimulatory molecule expression results, suggesting that the BECC molecules may be useful in future vaccine preparations.
Asunto(s)
Adyuvantes Inmunológicos/química , Descubrimiento de Drogas , Lípido A/biosíntesis , Lipopolisacáridos/química , Receptor Toll-Like 4/inmunología , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Línea Celular , Técnicas Químicas Combinatorias , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Escherichia coli/química , Humanos , Inmunomodulación , Interleucina-8/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Ligandos , Lípido A/análogos & derivados , Lípido A/química , Lípido A/inmunología , Lípido A/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Ratones , FN-kappa B/metabolismo , Receptor Toll-Like 4/agonistas , Factor de Necrosis Tumoral alfa/biosíntesis , Yersinia pestis/químicaRESUMEN
BACKGROUND: UV-exposures result in accumulation of genetic lesions that facilitate the development of skin cancer. Numerous pharmacologic agents are currently under development to both inhibit formation of DNA lesions and enhance repair. Drugs must be evaluated in vitro, currently performed in cell culture systems, before being tested on humans. Current systems do not account for the architecture and diverse cellularity of intact human skin. OBJECTIVE: To establish a novel, functionally viable, and reproducible in vitro skin organ culture system for studying the effects of various pharmacologic agents on DNA repair. METHODS: Human skin was obtained from neonatal foreskins. Intact skin punches derived from foreskins were cultured in vitro prior to exposure to UV-irradiation, and evaluated for DNA-damage using a DNA dot blot. Serial skin biopsies were obtained from patients with actinic keratoses treated with topical imiquimod. Expression of immune-stimulating and DNA repair genes was evaluated in ex vivo and in vitro samples. RESULTS: DNA dot blots revealed active repair of UV induced lesions in our in vitro skin organ culture. The photo-protective effect of sunscreen was detected, while imiquimod treatment did not enhance DNA repair in vitro. The DNA repair molecules XPA and XPF were up-regulated in the skin of imiquimod treated patients with actinic keratoses and imiquimod treated bone marrow-derived cell lines, but not keratinocytes. CONCLUSION: Our in vitro human skin organ culture model detected repair of UV-induced DNA lesions, and may be easily adapted to investigate various photo-protective drugs intended to prevent or treat skin cancer.