RESUMEN

Monophosphoryl lipid A (MPL®) is the first non-alum vaccine adjuvant to achieve widespread clinical and market acceptance, a remarkable achievement given that it is manufactured from a Salmonella enterica endotoxin. To understand how MPL® successfully balanced the dual mandate of vaccine design-low reactogenicity with high efficacy-clinical- and research-grade MPL was evaluated in human and mouse cell systems. Stimulatory dose response curves revealed that most preparations of MPL are much more active in mouse than in human cell systems, and that the limited efficacy observed in human cells correlated with TLR4 inhibitory activity that resulted in a partial agonist profile. Further analysis of the major components of MPL® adjuvant prepared synthetically identified two structural variants that functioned as competitive antagonists of human TLR4. A partial agonist profile could be recapitulated and manipulated by spiking synthetic agonists with synthetic antagonists to achieve a broad dose range over which TLR4 stimulation could be constrained below a desired threshold. This report thus identifies mixed agonist-antagonist activity as an additional mechanism by which MPL® adjuvant is detoxified, relative to its parental LPS, to render it safe for use in prophylactic vaccines.

Asunto(s)

Adyuvantes Inmunológicos/farmacología , Lípido A/análogos & derivados , Macrófagos/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Agonismo Parcial de Drogas , Humanos , Lípido A/farmacología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7 , Especificidad de la Especie , Células THP-1 , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo

RESUMEN

The Mfa1 fimbriae of the periodontal pathogen Porphyromonas gingivalis are involved in adhesion, including binding to synergistic species in oral biofilms. Mfa1 fimbriae are comprised of 5 proteins: the structural component Mfa1, the anchor Mfa2, and Mfa3-5 which constitute the fimbrial tip complex. Interactions among the Mfa proteins and the polymerization mechanism for Mfa1 are poorly understood. Here we show that Mfa3 can bind to Mfa1, 2, 4, and 5 in vitro, and may function as an adaptor protein interlinking other fimbrial subunits. Polymerization of Mfa1 is independent of Mfa3-5 and requires proteolytic processing mediated by the RgpA/B arginine gingipains of P. gingivalis. Both the N- and C- terminal regions of Mfa1 are necessary for polymerization; however, potential ß-strand disrupting amino acid substitutions in these regions do not impair Mfa1 polymerization. In contrast, substitution of hydrophobic amino acids with charged residues in either the N- or C- terminal domains yielded Mfa1 proteins that failed to polymerize. Collectively, these results indicate that Mfa3 serves as an adaptor protein between Mfa1 and other accessory fimbrial proteins. Mfa1 fimbrial polymerization is dependent on hydrophobicity in both the N- and C-terminal regions, indicative of an assembly mechanism involving the terminal regions forming a hydrophobic binding interface between Mfa1 subunits.

Asunto(s)

Proteínas Bacterianas/metabolismo , Infecciones por Bacteroidaceae/microbiología , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Enfermedades Periodontales/microbiología , Porphyromonas gingivalis/patogenicidad , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Biopelículas , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Proteínas Fimbrias/genética , Cisteína-Endopeptidasas Gingipaínas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Porphyromonas gingivalis/genética , Agregado de Proteínas , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Development of non-infectious subunit vaccines is hampered by a slow pipeline of new adjuvants to replace or enhance alum in part because expectations of safety are high. Transient vaccine side effects are not clinical priorities because they cause no lasting harm and vaccine development has appropriately been focused on avoidance of serious adverse events. As a result, surprisingly little is known about the extent to which side effects caused by a vaccine's reactogencicity are predictive of successful immunization outcomes. Recent clinical studies of pertussis and human papillomavirus vaccines adjuvanted with alum or the TLR4 agonist monophosphoryl lipid A can be used to advance understanding of the relationship between vaccine side effects and immunization outcomes.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Anticuerpos Antivirales/metabolismo , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/inmunología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Lípido A/análogos & derivados , Dolor/prevención & control , Infecciones por Papillomavirus/inmunología , Vacunas contra Papillomavirus/inmunología , Tos Ferina/inmunología , Adyuvantes Inmunológicos/efectos adversos , Compuestos de Alumbre/efectos adversos , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/efectos adversos , Humanos , Lípido A/administración & dosificación , Lípido A/farmacología , Dolor/etiología , Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus/efectos adversos , Receptor Toll-Like 4/agonistas , Resultado del Tratamiento , Vacunación , Tos Ferina/prevención & control

RESUMEN

Signaling by Toll-like receptor 4 (TLR4) is mediated by either of two adaptor proteins: myeloid differentiation marker 88 (MyD88) or Toll-interleukin-1 (IL-1) receptor (TIR) domain-containing adaptor inducing interferon-ß (TRIF). Whereas MyD88-mediated signaling leads to proinflammatory responses, TRIF-mediated signaling leads to less toxic immunostimulatory responses that are beneficial in boosting vaccine responses. The hypothesis that monophosphorylated lipid A structures act as TRIF-biased agonists of TLR4 offered a potential mechanism to explain their clinical value as vaccine adjuvants, but studies of TRIF-biased agonists have been contradictory. In experiments with mouse dendritic cells, we found that irrespective of the agonist used, TLR4 functioned as a TRIF-biased signaling system through a mechanism that depended on the autocrine and paracrine effects of type I interferons. The TLR4 agonist synthetic lipid A induced expression of TRIF-dependent genes at lower concentrations than were necessary to induce the expression of genes that depend on MyD88-mediated signaling. Blockade of type I interferon signaling selectively decreased the potency of lipid A (increased the concentration required) in inducing the expression of TRIF-dependent genes, thereby eliminating adaptor bias. These data may explain how high-potency TLR4 agonists can act as clinically useful vaccine adjuvants by selectively activating TRIF-dependent signaling events required for immunostimulation, without or only weakly activating potentially harmful MyD88-dependent inflammatory responses.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Regulación de la Expresión Génica , Interferón Tipo I/metabolismo , Lípido A/química , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Citocinas/metabolismo , Células Dendríticas/citología , Inflamación , Interferón beta/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal

RESUMEN

Synthetic forms of E. coli monophosphoryl lipid A (sMLA) weakly activate the MyD88 (myeloid differentiation primary response protein) branch of the bifurcated TLR4 (Toll-like receptor 4) signaling pathway, in contrast to diphosphoryl lipid A (sDLA), which is a strong activator of both branches of TLR4. sMLA's weak MyD88 signaling activity is apparent downstream of TLR4/MyD88 signaling as we show that sMLA, unlike sDLA, is unable to efficiently recruit the TNF receptor-associated factor 6 (TRAF6) to the Interleukin-1 receptor-associated kinase 1 (IRAK1). This reduced recruitment of TRAF6 explains MLA's lower MAPK (Mitogen Activated Protein Kinase) and NF-κB activity. As further tests of sMLA's ability to activate TLR4/Myeloid differentiation factor 2 (MD-2), we used the antibody MTS510 as an indicator for TLR4/MD-2 heterotetramer formation. Staining patterns with this antibody indicated that sMLA does not effectively drive heterotetramerization of TLR4/MD-2 when compared to sDLA. However, a F126A mutant of MD-2, which allows lipid A binding but interferes with TLR4/MD-2 heterotetramerization, revealed that while sMLA is unable to efficiently form TLR4/MD-2 heterotetramers, it still needs heterotetramer formation for the full extent of signaling it is able to achieve. Monophosphoryl lipid A's weak ability to form TLR4/MD-2 heterotetramers was not restricted to synthetic E. coli type because cells exposed to a biological preparation of S. minnesota monophosphoryl lipid A (MPLA) also showed reduced TLR4/MD-2 heterotetramer formation. The low potency with which sMLA and MPLA drive heterotetramerization of TLR4/MD-2 contributes to their weak MyD88 signaling activities.

Asunto(s)

Lípido A/análogos & derivados , Antígeno 96 de los Linfocitos/metabolismo , Multimerización de Proteína/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Animales , Células HEK293 , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Lípido A/farmacología , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Fosfatos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/metabolismo

RESUMEN

We previously showed that monophosphoryl lipid A (MLA) activates TLR4 in dendritic cells (DCs) in a Toll/IL-1R domain-containing adaptor inducing IFN-ß (TRIF)-biased manner: MLA produced from Salmonella minnesota Re595 induced signaling events and expression of gene products that were primarily TRIF dependent, whereas MyD88-dependent signaling was impaired. Moreover, when tested in TRIF-intact/MyD88-deficient DCs, synthetic MLA of the Escherichia coli chemotype (sMLA) showed the same activity as its diphosphoryl, inflammatory counterpart (synthetic diphosphoryl lipid A), indicating that TRIF-mediated signaling is fully induced by sMLA. Unexpectedly, we found that the transcript level of one proinflammatory cytokine was increased in sMLA-treated cells by MyD88 deficiency to the higher level induced by synthetic diphosphoryl lipid A, which suggested MyD88 may paradoxically help restrain proinflammatory signaling by TRIF-biased sMLA. In this article, we demonstrate that sMLA induces MyD88 recruitment to TLR4 and activates the anti-inflammatory lipid phosphatase SHIP1 in an MyD88-dependent manner. At the same time, MyD88-dependent signaling activity at the level of IL-1R-associated kinase 1 is markedly reduced. Increased SHIP1 activity is associated with reductions in sMLA-induced IκB kinase α/ß and IFN regulatory factor 3 activation and with restrained expression of their downstream targets, endothelin-1 and IFN-ß, respectively. Results of this study identify a pattern that is desirable in the context of vaccine adjuvant design: TRIF-biased sMLA can stimulate partial MyD88 activity, with MyD88-dependent SHIP1 helping to reduce proinflammatory signaling in DCs.

Asunto(s)

Adyuvantes Inmunológicos/fisiología , Células Dendríticas/inmunología , Mediadores de Inflamación/fisiología , Lípido A/análogos & derivados , Factor 88 de Diferenciación Mieloide/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Transducción de Señal/inmunología , Receptor Toll-Like 4/metabolismo , Adyuvantes Inmunológicos/antagonistas & inhibidores , Adyuvantes Inmunológicos/metabolismo , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/microbiología , Células de la Médula Ósea/patología , Células Dendríticas/microbiología , Células Dendríticas/patología , Escherichia coli/inmunología , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Inositol Polifosfato 5-Fosfatasas , Lípido A/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Salmonella/inmunología , Transducción de Señal/genética , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/fisiología

RESUMEN

TLR4 stimulation by lipopolysaccharide can cause both MAL/MyD88- and TRAM/TRIF (Toll IL-1 receptor domain-containing adaptor-inducing IFNbeta)-dependent signaling events. Monophosphoryl lipid A (MPLA), a low toxicity derivative of endotoxic lipopolysaccharide, enhances antibody responses, T cell expansion, and recall responses against antigens without causing excessive inflammatory side effects. Previously, we proposed that TRIF-biased activation of TLR4 by MPLA is responsible for its reduced toxicity while retaining potent adjuvant effects. However, some TRIF-associated genes, such as MCP-1, are only weakly expressed, and some MyD88-associated inflammatory and anti-inflammatory cytokines, such as tumor necrosis factor alpha and interleukin-10, are strongly activated after MPLA stimulation despite weak NF-kappaB but strong IRF3 activation. We now report that synthetic derivatives of MPLA retained TRIF bias as compared with synthetic diphosphoryl lipid A, indicating a change in a single phosphoryl group is sufficient for TRIF-biased TLR4 stimulation. We extend our previous observations by showing that sMLA induces strong p38 MAPK but weak JNK activation, resulting in high IP-10 (interferon-inducible protein 10), tumor necrosis factor alpha, and interleukin-10 but low MCP-1 transcript levels. Results of this study identify a novel biochemical mechanism for regulation of sMLA-induced gene expression.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular/fisiología , Adyuvantes Inmunológicos/farmacología , Células Dendríticas/metabolismo , Lípido A/análogos & derivados , Factor 88 de Diferenciación Mieloide/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Western Blotting , Médula Ósea/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Lípido A/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo

RESUMEN

The inflammatory toxicity of lipopolysaccharide (LPS), a component of bacterial cell walls, is driven by the adaptor proteins myeloid differentiation factor 88 (MyD88) and Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta (TRIF), which together mediate signaling by the endotoxin receptor Toll-like receptor 4 (TLR4). Monophosphoryl lipid A (MPLA) is a low-toxicity derivative of LPS with useful immunostimulatory properties, which is nearing regulatory approval for use as a human vaccine adjuvant. We report here that, in mice, the low toxicity of MPLA's adjuvant function is associated with a bias toward TRIF signaling, which we suggest is likely caused by the active suppression, rather than passive loss, of proinflammatory activity of this LPS derivative. This finding may have important implications for the development of future vaccine adjuvants.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adyuvantes Inmunológicos , Lípido A/análogos & derivados , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/toxicidad , Traslado Adoptivo , Animales , Citocinas/biosíntesis , Inmunización , Lípido A/administración & dosificación , Lípido A/inmunología , Lípido A/toxicidad , Lipopolisacáridos/inmunología , Activación de Linfocitos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovalbúmina/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Receptor Toll-Like 4/metabolismo

RESUMEN

Activated T cell death (ATCD) after peak clonal expansion is required for effective homeostasis of the immune system. Using a mouse model of T cell clonal expansion and contraction, we found that regulation of the proapoptotic kinase glycogen synthase kinase (GSK)-3beta plays a decisive role in determining the extent to which T cells are eliminated after activation. Involvement of GSK-3beta in ATCD was tested by measuring T cell survival after GSK-3beta inhibition, either ex vivo with chemical and pharmacological inhibitors or in vivo by retroviral expression of a dominant-negative form of GSK-3. We also measured amounts of inactivating phosphorylation of GSK-3beta (Ser9) in T cells primed in the presence or absence of LPS. Our results show that GSK-3beta activity is required for ATCD and that its inhibition promoted T cell survival. Adjuvant treatment in vivo maintained GSK-3beta (Ser9) phosphorylation in activated T cells, whereas with adjuvant-free stimulation it peaked and then decayed as the cells became susceptible to ATCD. We conclude that the duration of GSK-3beta inactivation determines activated T cell survival and that natural adjuvant stimulation decreases the severity of clonal contraction in part by keeping a critical proapoptotic regulatory factor, GSK-3beta, inactivated.

Asunto(s)

Adyuvantes Inmunológicos/fisiología , Apoptosis/inmunología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Femenino , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/efectos de los fármacos

RESUMEN

The viral infectivity factor gene (vif) of HIV-1 increases the infectivity of viral particles by inactivation of cellular anti-viral factors, and supports productive viral replication in primary human CD4 T cells and in certain non-permissive T cell lines. Here, we demonstrate that Vif also contributes to the arrest of HIV-1 infected cells in the G(2) phase of the cell cycle. Viruses deleted in Vif or Vpr induce less cell cycle arrest than wild-type virus, while cells infected with HIV-1 deleted in both Vif and Vpr have a cell cycle profile equivalent to that of uninfected cells. Furthermore, expression of Vif alone induces accumulation of cells in the G(2) phase of the cell cycle. These data demonstrate a novel role for Vif in cell cycle regulation and suggest that Vif and Vpr independently drive G(2) arrest in HIV-1 infected cells. Our results may have implications for the actions and interactions of key HIV-1 accessory proteins in AIDS pathogenesis.

Asunto(s)

Ciclo Celular , Productos del Gen vif/metabolismo , VIH-1/fisiología , Linfocitos T/citología , Linfocitos T/virología , Células Cultivadas , Regulación Viral de la Expresión Génica , Productos del Gen vif/genética , Productos del Gen vpr/genética , Productos del Gen vpr/metabolismo , VIH-1/genética , Humanos , Células Jurkat , Mutación , Linfocitos T/fisiología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana

RESUMEN

Immunological adjuvants increase the clonal burst size of antigen-specific T cell populations by mechanisms that remain incompletely understood. Using the DO11.10 adoptive transfer system to study peptide-stimulated T cell responses, we found that TLR agonist treatment increased the extent of cellular division undergone by responding T cells, but not by enough to explain the net increases in T cell yield that were achieved. Two novel analyses involving CFSE dye dilution analysis were used to characterize the shortfall, both of which were consistent with the idea that DO11.10 T cells are frequently lost during proliferation unless TLR agonists are present. T cell loss during clonal expansion was correlated with decreased levels of Bcl-2, but TLR agonists did not appear to afford protection by restoring levels of Bcl-2 or of cell surface IL-7Ralpha chain expression. TLR-mediated protection also failed to correlate with increased expression of Bcl-x or decreased expression of pro-apoptotic Bim. Our findings suggest that DO11.10 T cells stimulated by antigenic peptide in vivo divide well, but fail to accumulate efficiently unless TLR agonists are present.

Asunto(s)

Agregación Celular/inmunología , División Celular/inmunología , Péptidos/farmacología , Subgrupos de Linfocitos T/citología , Receptores Toll-Like/agonistas , Receptores Toll-Like/fisiología , Animales , Linfocitos T CD4-Positivos/fisiología , Agregación Celular/genética , Ciclo Celular/genética , Ciclo Celular/inmunología , División Celular/genética , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Células Clonales , Tejido Linfoide/citología , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Interleucina-7/biosíntesis , Receptores de Interleucina-7/genética