RESUMO
Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.
Assuntos
Vírus da Hepatite B da Marmota/imunologia , Hepatite B Crônica/veterinária , Imunidade Celular/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Interferon-alfa/uso terapêutico , Fígado/metabolismo , Transcrição Gênica , Animais , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Antivirais/metabolismo , Antivirais/uso terapêutico , Biomarcadores/sangue , Biomarcadores/metabolismo , Biópsia , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/genética , Fatores Imunológicos/metabolismo , Interferon-alfa/administração & dosagem , Interferon-alfa/genética , Interferon-alfa/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Fígado/imunologia , Fígado/patologia , Fígado/virologia , Masculino , Marmota , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Carga Viral/efeitos dos fármacosRESUMO
When they infect Escherichia coli cells, the filamentous phages IF1 and fd first interact with a pilus and then target TolA as their common receptor. They use the domains N2 and N1 of their gene-3-proteins (G3P) for these interactions but differ in the mechanism of infection. In G3P of phage IF1, N1 and N2 are independent modules that are permanently binding-active. G3P of phage fd is usually in a closed state in which N1 and N2 are tightly associated. The TolA binding site is thus inaccessible and the phage incompetent for infection. Partial unfolding and prolyl isomerization must occur to abolish the domain interactions and expose the TolA binding site. This complex mechanism of phage fd could be changed to the simple infection mechanism of phage IF1 by reprogramming its G3P following physicochemical rules of protein stability. The redesigned phage fd was robust and as infectious as wild-type phage fd.
Assuntos
Escherichia coli/virologia , Inovirus/fisiologia , Receptores Virais/metabolismo , Proteínas Virais/metabolismo , Ligação Viral , Substituição de Aminoácidos/genética , Sítios de Ligação , Inovirus/crescimento & desenvolvimento , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Virais/genéticaRESUMO
OBJECTIVE: Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor α (TNFα) or interleukin-6 (IL-6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNFα and IL-17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo. METHODS: Cultures of human fibroblast-like synoviocytes (FLS) were stimulated with TNFα, IL-17, or a combination of both. Single/combined neutralizing antibodies against TNFα and IL-17 were used to examine in vitro cytokine responses and in vivo development of arthritis and bone and cartilage destruction in TNFα-transgenic mice. Bispecific anti-TNFα/IL-17 antibodies were designed, and their potential to block cytokine responses in human FLS was tested. RESULTS: TNFα and IL-17 had additive/synergistic effects in promoting production of IL-6, IL-8, and granulocyte colony-stimulating factor, as well as matrix metalloproteinases, in FLS. Bispecific anti-TNFα/IL-17 antibodies showed superior efficacy in blocking cytokine and chemokine responses in vitro. Furthermore, dual versus single inhibition of both cytokines using neutralizing antibodies was more effective in inhibiting the development of inflammation and bone and cartilage destruction in arthritic mice. CONCLUSION: Combined blockade of TNFα and IL-17 was more effective than single blockade in inhibiting cytokine, chemokine, and matrix enzyme responses from human mesenchymal cells and in blocking tissue destruction associated with arthritis, and additionally showed a positive impact on rebalance of bone homeostasis. Bispecific anti-TNFα/IL-17 antibodies may have superior efficacy in the treatment of arthritis and may overcome the limited therapeutic responses obtained with single cytokine neutralization.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Interleucina-17/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/imunologia , Antirreumáticos/imunologia , Artrite Reumatoide/patologia , Células Cultivadas , Modelos Animais de Doenças , Sinergismo Farmacológico , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Técnicas In Vitro , Interleucina-17/imunologia , Interleucina-17/farmacologia , Interleucina-8/metabolismo , Metaloproteases/metabolismo , Camundongos , Camundongos Transgênicos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The filamentous phage fd uses its gene 3 protein (G3P) to target Escherichia coli cells in a two-step process. First, the N2 domain of G3P attaches to an F pilus, and then the N1 domain binds to TolA-C. N1 and N2 are tightly associated, rendering the phage robust but noninfectious because the binding site for TolA-C is buried at the domain interface. Binding of N2 to the F pilus initiates partial unfolding, domain disassembly, and prolyl cis-to-trans isomerization in the hinge between N1 and N2. This activates the phage, and trans-Pro213 maintains this state long enough for N1 to reach TolA-C. Phage IF1 targets I pili, and its G3P contains also an N1 domain and an N2 domain. The pilus-binding N2 domains of the phages IF1 and fd are unrelated, and the N1 domains share a 31% sequence identity. We show that N2 of phage IF1 mediates binding to the I pilus, and that N1 targets TolA. Crystallographic and NMR analyses of the complex between N1 and TolA-C indicate that phage IF1 interacts with the same site on TolA-C as phage fd. In IF1-G3P, N1 and N2 are independently folding units, however, and the TolA binding site on N1 is permanently accessible. Activation by unfolding and prolyl isomerization, as in the case of phage fd, is not observed. In IF1-G3P, the absence of stabilizing domain interactions is compensated for by a strong increase in the stabilities of the individual domains. Apparently, these closely related filamentous phages evolved different mechanisms to reconcile robustness with high infectivity.
Assuntos
Bacteriófago M13/genética , Bacteriófago M13/patogenicidade , Colífagos/genética , Colífagos/patogenicidade , Escherichia coli/virologia , Inovirus/genética , Inovirus/patogenicidade , Sequência de Aminoácidos , Bacteriófago M13/fisiologia , Sítios de Ligação , Colífagos/fisiologia , Cristalografia por Raios X , Escherichia coli/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Genes Virais , Inovirus/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/fisiologia , Virulência/genética , Virulência/fisiologiaRESUMO
In the N2 domain of the gene-3-protein of phage fd, two consecutive beta-strands are connected by a mobile loop of seven residues (157-163). The stability of this loop is low, and the Asp160-Pro161 bond at its tip shows conformational heterogeneity with 90% being in the cis and 10% in the trans form. The refolding kinetics of N2 are complex because the molecules with cis or trans isomers at Pro161 both fold to native-like conformations, albeit with different rates. We employed consensus design to shorten the seven-residue irregular loop around Pro161 to a four-residue type I' turn without a proline. This increased the conformational stability of N2 by almost 10 kJ mol(-1) and abolished the complexity of the folding kinetics. Turn sequences obtained from in vitro selections for increased stability strongly resembled those derived from the consensus design. Two other type I' turns of N2 could also be stabilized by consensus design. For all three turns, the gain in stability originates from an increase in the rate of refolding. The turns form native-like structures early during refolding and thus stabilize the folding transition state. The crystal structure of the variant with all three stabilized turns confirms that the 157-163 loop was in fact shortened to a type I' turn and that the other turns maintained their type I' conformation after sequence optimization.