RESUMEN
In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4+ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After â¼3 mo, the decay slope changes, and CD4+ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5' or 3' end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.
Asunto(s)
Antirretrovirales/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Provirus/efectos de los fármacos , Virión/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , ADN Viral/efectos de los fármacos , Humanos , Estudios Longitudinales , Carga Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The human immunodeficiency virus (HIV) reservoir is responsible for persistent viral infection, and a small number of mosaic latent cellular reservoirs promote viral rebound upon antiretroviral therapy interruption, which is the major obstacle to a cure. However, markers that determine effective therapy and viral rebound posttreatment interruption remain unclear. In this study, we comprehensively and longitudinally tracked dynamic decay of cell-associated viral RNA/DNA in systemic and lymphoid tissues in simian immunodeficiency virus (SIV)-infected rhesus macaques on prolonged combined antiretroviral therapy (cART) and evaluated predictors of viral rebound after treatment cessation. The results showed that suppressive ART substantially reduced plasma SIV RNA, cell-associated unspliced, and multiply spliced SIV RNA to undetectable levels, yet viral DNA remained detectable in systemic tissues and lymphoid compartments throughout cART. Intriguingly, a rapid increase of integrated proviral DNA in peripheral mononuclear cells was detected once treatment was withdrawn, accompanied by the emergence of detectable plasma viral load. Notably, the increase of peripheral proviral DNA after treatment interruption correlated with the emergence and degree of viral rebound. These findings suggest that measuring total viral DNA in SIV infection may be a relatively simple surrogate marker of reservoir size and may predict viral rebound after treatment interruption and inform treatment strategies.IMPORTANCE Viral reservoirs are involved in persistent HIV infection, and a small number of mosaic latent cellular reservoirs promote viral rebound upon analytical treatment interruption, which is the major obstacle to a cure. However, early indicators that can predict resurgence of viremia after treatment interruption may aid treatment decisions in people living with HIV. Utilizing the rhesus macaque model, we demonstrated that increased proviral DNA in peripheral cells after treatment interruption, rather than levels of proviral DNA, was a useful marker to predict the emergence and degree of viral rebound after treatment interruption, providing a rapid approach for monitoring HIV rebound and informing decisions.
Asunto(s)
ADN Viral/metabolismo , Provirus/fisiología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Activación Viral , Animales , Antirretrovirales/uso terapéutico , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/virología , ADN Viral/efectos de los fármacos , Leucocitos Mononucleares/virología , Ganglios Linfáticos/virología , Macaca mulatta , Provirus/efectos de los fármacos , ARN Viral/sangre , ARN Viral/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Carga Viral/efectos de los fármacos , Viremia/tratamiento farmacológico , Viremia/virologíaRESUMEN
Drug resistance in viruses represents one of the major challenges of healthcare. As part of an effort to provide a treatment that avoids the possibility of drug resistance, we discovered a novel mechanism of action (MOA) and specific compounds to treat all nine human herpesviruses and animal herpesviruses. The novel MOA targets the pressurized genome state in a viral capsid, "turns off" capsid pressure, and blocks viral genome ejection into a cell nucleus, preventing viral replication. This work serves as a proof-of-concept to demonstrate the feasibility of a new antiviral target-suppressing pressure-driven viral genome ejection-that is likely impervious to developing drug resistance. This pivotal finding presents a platform for discovery of a new class of broad-spectrum treatments for herpesviruses and other viral infections with genome-pressure-dependent replication. A biophysical approach to antiviral treatment such as this is also a vital strategy to prevent the spread of emerging viruses where vaccine development is challenged by high mutation rates or other evasion mechanisms.
Asunto(s)
Antivirales/farmacología , Cápside/efectos de los fármacos , ADN Viral/efectos de los fármacos , Infecciones por Herpesviridae , Herpesviridae/efectos de los fármacos , Animales , Cápside/fisiología , Chlorocebus aethiops , ADN Viral/fisiología , Herpesviridae/fisiología , Humanos , Ratones , Prueba de Estudio Conceptual , Ratas , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND AND AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a pivotal role in the establishment and persistence of HBV infection. Understanding the turnover time of preexisting cccDNA pools would be helpful in designing strategies to clear HBV by fully blocking the de novo generation of cccDNA. APPROACH AND RESULTS: In this study, we retrospectively monitored the emergence and reversion of the rtM204I/V mutant, a signature lamivudine resistance (LAMR ) mutation serving as a biomarker of cccDNA turnover in liver biopsies and longitudinal serum samples from two clinical trials. Methodologies were optimized to differentially isolate and sequence HBV virion DNA, cccDNA, and HBV RNA from clinical samples. A strong correlation was observed between LAMR composition of cccDNA with that of serum and intrahepatic HBV RNA in paired liver and serum samples (r = 0.96 and 0.90, respectively), suggesting that serum HBV RNA can serve as a surrogate marker of cccDNA genetic composition when liver biopsies are unavailable. LAMR mutations emerged and increased from undetectable to 40%-90% within 16-28 weeks in serum HBV RNA from telbivudine-treated patients experiencing virological breakthrough. Similarly, in lamivudine-resistant patients who switched to interferon therapy, serum HBV-RNA population bearing 100% LAMR mutations fully reversed back to wild type within 24-48 weeks. CONCLUSIONS: The genetic composition dynamics of serum HBV RNA and biopsy cccDNA in treated HBV patients indicates that cccDNA turnover occurs relatively rapidly (several months), offering a possibility of HBV cure with finite therapy through completely blocking cccDNA replenishment.
Asunto(s)
Antivirales/uso terapéutico , ADN Circular/efectos de los fármacos , ADN Viral/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Lamivudine/uso terapéutico , Adolescente , Adulto , Anciano , Biomarcadores/sangre , ADN Viral/sangre , Femenino , Antígenos del Núcleo de la Hepatitis B/sangre , Antígenos e de la Hepatitis B/sangre , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Hepatitis B Crónica/virología , Humanos , Hígado/efectos de los fármacos , Hígado/virología , Masculino , Persona de Mediana Edad , Mutación , Estudios Retrospectivos , Resultado del Tratamiento , Carga Viral , Adulto JovenRESUMEN
Interleukin-6 (IL-6) binds to the IL-6 receptor (IL-6R) subunit, related to autoimmune diseases and cytokine storm in COVID-19. In this study, we performed systematic evolution of ligands by exponential enrichment and identified a novel RNA aptamer. This RNA aptamer not only bound to IL-6R with a dissociation constant of 200 n m, but also inhibited the interaction of IL-6R with IL-6.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Interleucina-6/antagonistas & inhibidores , Receptores de Interleucina-6/antagonistas & inhibidores , Aptámeros de Nucleótidos/química , Secuencia de Bases , COVID-19/complicaciones , Síndrome de Liberación de Citoquinas/etiología , ADN Viral/efectos de los fármacos , Humanos , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Técnica SELEX de Producción de AptámerosRESUMEN
Capsid assembly is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the core protein. Core is a potential target for new antiviral therapies, the capsid assembly modulators (CAMs). JNJ-56136379 (JNJ-6379) is a novel and potent CAM currently in phase II trials. We evaluated the mechanisms of action (MOAs) and antiviral properties of JNJ-6379 in vitro Size exclusion chromatography and electron microscopy studies demonstrated that JNJ-6379 induced the formation of morphologically intact viral capsids devoid of genomic material (primary MOA). JNJ-6379 accelerated the rate and extent of HBV capsid assembly in vitro JNJ-6379 specifically and potently inhibited HBV replication; its median 50% effective concentration (EC50) was 54 nM (HepG2.117 cells). In HBV-infected primary human hepatocytes (PHHs), JNJ-6379, when added with the viral inoculum, dose-dependently reduced extracellular HBV DNA levels (median EC50 of 93 nM) and prevented covalently closed circular DNA (cccDNA) formation, leading to a dose-dependent reduction of intracellular HBV RNA levels (median EC50 of 876 nM) and reduced antigen levels (secondary MOA). Adding JNJ-6379 to PHHs 4 or 5 days postinfection reduced extracellular HBV DNA and did not prevent cccDNA formation. Time-of-addition PHH studies revealed that JNJ-6379 most likely interfered with postentry processes. Collectively, these data demonstrate that JNJ-6379 has dual MOAs in the early and late steps of the HBV life cycle, which is different from the MOA of nucleos(t)ide analogues. JNJ-6379 is in development for chronic hepatitis B treatment and may translate into higher HBV functional cure rates.
Asunto(s)
Antivirales/farmacología , Cápside/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Compuestos Orgánicos/farmacología , Cápside/ultraestructura , Proteínas de la Cápside/metabolismo , Línea Celular , Replicación del ADN/efectos de los fármacos , ADN Viral/biosíntesis , ADN Viral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Hepatitis B/tratamiento farmacológico , Hepatitis B/virología , Virus de la Hepatitis B/ultraestructura , Hepatocitos/virología , Humanos , Pruebas de Sensibilidad Microbiana , Cultivo Primario de Células , Replicación Viral/efectos de los fármacosRESUMEN
APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination.IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.
Asunto(s)
Aminohidrolasas/metabolismo , Aminohidrolasas/farmacología , Antivirales/farmacología , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Desaminasas APOBEC/metabolismo , Aminohidrolasas/química , Aminohidrolasas/genética , Línea Celular , ADN Viral/efectos de los fármacos , ADN Viral/metabolismo , VIH-1/genética , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas con Motivos de Reconocimiento de ARN , Proteínas de Unión al ARN/química , Transcripción Reversa , ViriónRESUMEN
In actual clinical practice, infinite nucleos(t)ide analogues (NAs) treatment for chronic hepatitis B virus (HBV) infection is unrealistic. The most commonly used endpoint is suppression of HBV DNA to undetectable levels with normalization of alanine aminotransferase. However, this criterion for cessation of treatment is associated with various incidences of virological and clinical relapse. Recent studies suggest that decreasing the hepatitis B surface antigen (HBsAg) level at the end of treatment (EOT) to an appropriate cut-off value appears to be a practicable and attainable cessation criterion. We performed a systematic review to explore the optimal cut-off value of HBsAg at EOT for the cessation of NAs treatment. Eleven studies with 1,716 patients were included in this review. When the HBsAg levels at EOT were <100 IU/mL and >100 IU/mL, the respective off-therapy virological relapse rates were 9.1%-19.6% (range) and 31.4%-86.8% (range) at ≥12 months off therapy, regardless of hepatitis B e antigen (HBeAg) status; the respective off-therapy clinical relapse rates were 15.4%-29.4% (range) and 48.1%-63.6% (range) at ≥12 months off therapy, regardless of HBeAg status; and the respective off-therapy HBsAg loss rates were 21.1%-58.8% (range) and 3.3%-7.4% (range) for HBeAg-negative patients at ≥39 months off therapy. Conclusion: Cessation of long-term NAs therapy before HBsAg seroclearance in patients with chronic hepatitis B is a feasible alternative to indefinite treatment. An HBsAg level <100 IU/mL at EOT seems to be a useful marker for deciding when to discontinue NAs therapy. However, regular monitoring is required after the cessation of NAs treatment, and long-term outcomes must be further evaluated.
Asunto(s)
Antivirales/uso terapéutico , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/inmunología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/etnología , Adulto , Biomarcadores , China , ADN Viral/efectos de los fármacos , Femenino , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Recurrencia , Medición de Riesgo , Privación de TratamientoRESUMEN
The design and synthesis of a number of new imidazo[4,5-b]pyridines is described. The heterocyclic scaffold possesses 6-chloro- or 5,6-dichloro-substitution and bears various 2-alkylamino-methyl or ethyl groups. The corresponding N1 and N3-tosylates are also presented. The anti-HBV activity of the compounds was evaluated in HBV infectious system at the level of HBV rcDNA secretion and CC50, EC50 and selectivity index values were determined. The tosylates showed low antiviral potency and relatively high cytotoxicity, on the contrary, a number of 2,5 and/or-6-substituted imidazopyridines, mainly those belonging to the 6-chloroimidazo[4,5-b]pyridine series, were endowed with a very interesting profile and were further investigated. The most promising among them, along with the reduction of the secreted HBV rcDNA, also caused a reduction in HBV cccDNA and pgRNA levels, with a concomitant accumulation of the intracellular encapsidated rcDNA. Surprisingly, the most active 2-diethylaminoethyl-substituted derivative (21d), was highly competitive to interferon.
Asunto(s)
Antivirales/farmacología , Diseño de Fármacos , Virus de la Hepatitis B/efectos de los fármacos , Imidazoles/farmacología , Piridinas/farmacología , Antivirales/síntesis química , Antivirales/química , ADN Viral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Imidazoles/síntesis química , Imidazoles/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
The objective of this study was to investigate the efficacy and potential side-effects of nucleotide/nucleoside analogues and hepatitis B immunoglobulin injection of newborns in blocking mother-to-child transmission of hepatitis B virus in the middle and late pregnancy period. 238 cases of enrolled pregnant women were divided into the Telbivudine group, the Tenofovir group, the Lamivudine group, and the hepatitis B immunoglobulin (HBIG) group. Enrolled patients received corresponding therapies. Clinical and laboratory data were collected. Results showed that the levels of HBV DNA of the enrolled pregnant women in the Telbivudine, Tenofovir, and Lamivudine groups decreased rapidly after 12 weeks of drug intervention compared with those in the control. HBsAg positive rate in newborns and in children 24 weeks after birth was 0/60, 0/60, 0/60, 3/30, and 11/28 in the Telbivudine, Tenofovir, Lamivudine, HBIG, and control groups, respectively. No significant side-effects were identified after following up to 12 months after birth. Our results show that routine HBV vaccine plus HBIG injections is insufficient in blocking mother-to-child HBV transmission. Administration of nucleotide/nucleoside analogues or HBIG at pregnancy is suggested to maximize the blocking of vertical HBV transmission.
Asunto(s)
Antivirales/administración & dosificación , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/transmisión , Inmunoglobulinas/administración & dosificación , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Antivirales/farmacología , Estudios de Casos y Controles , China , ADN Viral/efectos de los fármacos , ADN Viral/genética , Esquema de Medicación , Femenino , Edad Gestacional , Hepatitis B/prevención & control , Hepatitis B/virología , Antígenos de la Hepatitis B/sangre , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Humanos , Inmunoglobulinas/farmacología , Recién Nacido , Lamivudine/administración & dosificación , Lamivudine/farmacología , Embarazo , Telbivudina/administración & dosificación , Telbivudina/farmacología , Tenofovir/administración & dosificación , Tenofovir/farmacología , Resultado del TratamientoRESUMEN
BACKGROUND: Type III interferons (IFNs) (λ1-3) activate similar signaling cascades as type I IFNs (α and ß) via different receptors. Since IFN-α and lymphotoxin-ß activate cytosine deamination and subsequent purging of nuclear hepatitis B virus (HBV) DNA, we investigated whether IFN-ß and -λ may also induce these antiviral effects in differentiated HBV-infected hepatocytes. METHODS: After determining the biological activity of IFN-α2, -ß1, -λ1, and -λ2 in differentiated hepatocytes, their antiviral effects were analyzed in HBV-infected primary human hepatocytes and HepaRG cells. RESULTS: Type I and III IFNs reduced nuclear open-circle DNA and covalently closed circular DNA (cccDNA) levels in HBV-infected cells. IFN-ß and -λ were at least as efficient as IFN-α. Differential DNA-denaturing polymerase chain reaction and sequencing analysis revealed G-to-A sequence alterations of HBV cccDNA in IFN-α, -ß, and -λ-treated liver cells indicating deamination. All IFNs induced apolipoprotein B messenger RNA-editing enzyme-catalytic polypeptide-like (APOBEC) deaminases 3A and 3G within 24 hours of treatment, but IFN-ß and -λ induced longer-lasting expression of APOBEC deaminases in comparison to IFN-α. CONCLUSIONS: IFN-ß, IFN-λ1, and IFN-λ2 induce cccDNA deamination and degradation at least as efficiently as IFN-α, indicating that these antiviral cytokines are interesting candidates for the design of new therapeutic strategies aiming at cccDNA reduction and HBV cure.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/tratamiento farmacológico , Interferón Tipo I/farmacología , Interferones/farmacología , Células Cultivadas , Citocinas/inmunología , ADN Circular/efectos de los fármacos , ADN Viral/efectos de los fármacos , Hepatitis B/virología , Virus de la Hepatitis B/inmunología , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Interferón-alfa/inmunología , Interferón beta/inmunología , Interferones/inmunología , Interferón lambdaRESUMEN
BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is sensitive to interferon (IFN)-based therapy, whereas hepatitis B virus (HBV) infection is not. It is unclear whether HBV escapes detection by the IFN-mediated immune response or actively suppresses it. Moreover, little is known on how HBV and HCV influence each other in coinfected cells. We investigated interactions between HBV and the IFN-mediated immune response using HepaRG cells and primary human hepatocytes (PHHs). We analyzed the effects of HBV on HCV replication, and vice versa, at the single-cell level. METHODS: PHHs were isolated from liver resection tissues from HBV-, HCV-, and human immunodeficiency virus-negative patients. Differentiated HepaRG cells overexpressing the HBV receptor sodium taurocholate cotransporting polypeptide (dHepaRGNTCP) and PHHs were infected with HBV. Huh7.5 cells were transfected with circular HBV DNA genomes resembling viral covalently closed circular DNA (cccDNA), and subsequently infected with HCV; this served as a model of HBV and HCV coinfection. Cells were incubated with IFN inducers, or IFNs, and antiviral response and viral replication were analyzed by immune fluorescence, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assays, and flow cytometry. RESULTS: HBV infection of dHepaRGNTCP cells and PHHs neither activated nor inhibited signaling via pattern recognition receptors. Incubation of dHepaRGNTCP cells and PHHs with IFN had little effect on HBV replication or levels of cccDNA. HBV infection of these cells did not inhibit JAK-STAT signaling or up-regulation of IFN-stimulated genes. In coinfected cells, HBV did not prevent IFN-induced suppression of HCV replication. CONCLUSIONS: In dHepaRGNTCP cells and PHHs, HBV evades the induction of IFN and IFN-induced antiviral effects. HBV infection does not rescue HCV from the IFN-mediated response.
Asunto(s)
Antivirales/farmacología , Hepacivirus/inmunología , Virus de la Hepatitis B/inmunología , Hepatocitos/inmunología , Inmunidad Innata/inmunología , Interferones/farmacología , Coinfección/tratamiento farmacológico , Coinfección/inmunología , Coinfección/virología , ADN Viral/efectos de los fármacos , ADN Viral/inmunología , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepatitis B/tratamiento farmacológico , Hepatitis B/inmunología , Hepatitis B/virología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/inmunología , Hepatitis C/virología , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Hígado/citología , Hígado/inmunología , Hígado/virología , Replicación Viral/efectos de los fármacosRESUMEN
Chronic hepatitis B affects over 300 million people who are at risk of developing liver cancer. The basis for the persistence of hepatitis B virus (HBV) in hepatocytes, even in the presence of available antiviral therapies, lies in the accumulation of covalently closed circular DNA (cccDNA) in nuclei of infected cells. While methods for cccDNA quantification from liver biopsy specimens and cell lines expressing the virus are known, information about cccDNA formation, stability, and turnover is lacking. In particular, little is known about the fate of cccDNA during cell division. To fill the gaps in knowledge concerning cccDNA biology, we have developed a fluorescence imaging in situ hybridization (FISH)-based assay for the detection of duck hepatitis B virus (DHBV) cccDNA and HBV nuclear DNA in established cell lines. Using FISH, we determined the distribution of cccDNA under conditions mimicking chronic infections with and without antiviral therapy, which prevents de novo viral replication. Our results showed that the copy numbers of viral nuclear DNA can vary by as much as 1.8 orders of magnitude among individual cells and that antiviral therapy leads to a reduction in nuclear DNA in a manner consistent with symmetrical distribution of viral DNA to daughter cells.IMPORTANCE A mechanistic understanding of the stability of HBV cccDNA in the presence of antiviral therapy and during cell division induced by immune-mediated lysis of infected hepatocytes will be critical for the future design of curative antiviral therapies against chronic hepatitis B. Current knowledge about cccDNA stability was largely derived from quantitative analyses of cccDNA levels present in liver samples, and little was known about the fate of cccDNA in individual cells. The development of a FISH-based assay for cccDNA tracking provided the first insights into the fate of DHBV cccDNA and nuclear HBV DNA under conditions mimicking antiviral therapy.
Asunto(s)
ADN Circular/metabolismo , Virus de la Hepatitis B del Pato/genética , Virus de la Hepatitis B/genética , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , División Celular/genética , Replicación del ADN/efectos de los fármacos , ADN Circular/aislamiento & purificación , ADN Viral/efectos de los fármacos , ADN Viral/metabolismo , Hepatitis B Crónica/tratamiento farmacológico , Hepatocitos/virología , Hibridación Fluorescente in Situ/métodos , Replicación ViralRESUMEN
Children with chronic hepatitis B (CHB) represent an area of unmet medical need, attributed to increased lifetime risk of CHB sequelae and limited therapeutic options compared with adult CHB patients. The PEG-B-ACTIVE (NCT01519960) phase III study evaluated peginterferon (PegIFN) alfa-2a treatment in children aged 3 to <18 years with CHB. A total of 161 hepatitis B e antigen (HBeAg)-positive immune-active patients without advanced fibrosis (AF)/cirrhosis were randomized (2:1) to PegIFN alfa-2a (Group A, n = 101) or no treatment (Group B, n = 50); patients with AF were assigned to PegIFN alfa-2a (Group C, n = 10). PegIFN alfa-2a was administered for 48 weeks by body surface area (BSA) category, based on 180 µg/1.73 m2 . HBeAg seroconversion rates at 24 weeks posttreatment were significantly higher in Group A (25.7% vs. 6%; P = 0.0043), as were the rates of hepatitis B surface antigen (HBsAg) clearance (8.9% vs. 0%; P = 0.03), hepatitis B virus (HBV) DNA <2,000 IU/mL (28.7% vs. 2.0%; P < 0.001) or undetectable (16.8% vs. 2.0%; P = 0.0069), and alanine aminotransferase (ALT) normalization (51.5% vs. 12%; P < 0.001). Safety, including incidence of ALT flares and neutropenia, was comparable to the established PegIFN alfa-2a profile in HBV-infected adults or hepatitis C virus-infected children. Changes in growth parameters were minimal during treatment and comparable to those in untreated patients. Safety and efficacy outcomes in Group C were in line with Group A. Conclusion: PegIFN alfa-2a treatment of children in the immune-active phase of CHB was efficacious and well tolerated, and associated with higher incidence of HBsAg clearance than in adults. This represents an important advance to the treatment options for children with CHB.
Asunto(s)
Antivirales/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Polietilenglicoles/uso terapéutico , Adolescente , Antivirales/efectos adversos , Niño , Preescolar , ADN Viral/efectos de los fármacos , Femenino , Antígenos e de la Hepatitis B , Virus de la Hepatitis B , Humanos , Interferón-alfa/efectos adversos , Masculino , Polietilenglicoles/efectos adversos , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/uso terapéutico , Seroconversión/efectos de los fármacos , Resultado del TratamientoRESUMEN
Hepatitis B virus (HBV) is a global health problem requiring more efficient and better tolerated anti-HBV agent. In this paper, a series of novel 2'-deoxy-2'-fluoro-2'-C-methyl-ß-d-arabinofuranosyl 8-azanebularine analogues (1 and 2a) and N4-substituted 8-azaadenosine derivatives (2b-g) were designed, synthesized and screened for in vitro anti-HBV activity. Two concise and practical synthetic routes were developed toward the structural motif construction of 2'-deoxy-2'-fluoro-2'-C-methyl-ß-d-arabinofuranosyl 8-azainosine from the ribonolactone 3 under mild conditions. The in vitro anti-HBV screening results showed that these 8-azanebularine analogues had a significant inhibitory effect on the expression of HBV antigens and HBV DNA at a concentration of 20⯵M. Among them, halogen-substituted 8-azaadenosine derivative 2g displayed activities comparable to that of 3TC. In particular, 2g retained excellent activity against lamivudine-resistant HBV mutants.
Asunto(s)
Antivirales/farmacología , Diseño de Fármacos , Virus de la Hepatitis B/efectos de los fármacos , Nucleósidos de Purina/farmacología , Ribonucleósidos/farmacología , Antivirales/síntesis química , Antivirales/química , ADN Viral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/química , Ribonucleósidos/síntesis química , Ribonucleósidos/química , Relación Estructura-ActividadRESUMEN
To study the efficacy of a polyoxometalate, Cs2 K4 Na[SiW9 Nb3 O40 ]·H2 O, as an antiviral treatment in HBV transgenic mice. HBV transgenic mice were treated with Cs2 K4 Na[SiW9 Nb3 O40 ]·H2 O by intragastric administration. Adefovir and distilled water were administered as controls. Serum HBV DNA, liver HBV RNA levels were measured by quantitative RT-PCR. Serum HBsAg levels were measured by ELISA. The hepatitis B virus surface antigen (HBsAg) in liver cells was detected by immunohistochemistry (IHC). Pathological changes in the liver tissues were also observed by light and electron microscopy. Cs2 K4 Na[SiW9 Nb3 O40 ]·H2 O significantly decreased serum HBsAg and HBV DNA levels. Cs2 K4 Na[SiW9 Nb3 O40 ]·H2 O resulted in a 98% decrease in serum HBV DNA at 28 days, from 4.3 log10 copies/ml at baseline to 2.5 log10 copies/ml after treatment, and the inhibition rate of HBV DNA was higher than ADV at the same dose. The HBV replication levels in each group slightly increased at 7 days after withdrawal, but rebounded slightly more in the Cs2 K4 Na[SiW9 Nb3 O40 ]·H2 O treatment group compared to the H2 O control group (p < .05). There were no differences in HBV RNA levels. No significant differences were observed in the pathology, but there were decreased HBsAg levels in the Cs2 K4 Na[SiW9 Nb3 O40 ]·H2 O-treated group compared to the control group. The results demonstrated that Cs2 K4 Na[SiW9 Nb3 O40 ]·H2 O displayed potent anti-HBV activity in HBV transgenic mice and supported for future clinic study.
Asunto(s)
Antivirales/administración & dosificación , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/tratamiento farmacológico , Compuestos de Tungsteno/administración & dosificación , Animales , Antivirales/farmacología , ADN Viral/efectos de los fármacos , ADN Viral/genética , Modelos Animales de Enfermedad , Femenino , Hepatitis B/genética , Hepatitis B/virología , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Distribución Aleatoria , Compuestos de Tungsteno/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Pegylated interferon-alpha (pIFN-α) is suggested to lower human immunodeficiency virus type-1 (HIV-1) DNA load in antiretroviral therapy (ART)-treated patients. We studied kinetics of HIV-1 DNA levels in 40 HIV-1/hepatitis C virus (HCV) coinfected patients, treated with pIFN-α for HCV and categorized into 3 groups according to start of ART: chronic HIV-1 infection (n = 22), acute HIV-1 infection (n = 8), no-ART (n = 10). Total HIV-1 DNA levels in 247 peripheral blood mononuclear cell samples were stable before, during, and after pIFN-α treatment in all groups. Our results question the benefit of pIFN-α as an immunotherapeutic agent for reducing the HIV-1 reservoir.
Asunto(s)
Coinfección/tratamiento farmacológico , ADN Viral/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Hepatitis C Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Carga Viral/efectos de los fármacos , Adulto , Antivirales/uso terapéutico , Coinfección/virología , Femenino , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/virología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
BACKGROUND: Plasma Epstein-Barr virus (pEBV) DNA and fluorodeoxyglucose positron emission (PET) reflect tumour burden in advanced NPC. This study hypothesised that a dual endpoint based on assessing pEBV DNA clearance and PET response could predict early drug response. METHODS: Eligible patients underwent a computed tomography (CT) scan and dual PET-CT at baseline, a PET-CT at 4 weeks, and then a CT scan at 10 weeks after starting palliative or induction chemotherapy. Plasma EBV DNA clearance was determined. RESULTS: Fifty-eight out of 70 enrolled patients completed all imaging and 50/58 had falling pEBV DNA level, which allowed calculation of the clearance. At a median follow-up of 29.1 months, the dual endpoint (pEBV DNA clearance ≤ 10 days and > 50% drop in sum of SUVmax of target lesions) was an independent indicator of overall survival (hazard ratio (HR) = 0.135, 95% CI = 0.039 to 0.466, p = 0.0015) and progression-free survival (HR = 0.136, 95% CI = 0.048 to 0.385, p = 0002). This dual endpoint could predict subsequent response by Response Evaluation Criteria In Solid Tumours (RECIST) criteria at 10 weeks after chemotherapy. CONCLUSIONS: Early PET-CT response and pEBV DNA clearance could predict survival and subsequent response. This dual endpoint is an innovative tool for assessing early drug response.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , ADN Viral/sangre , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/diagnóstico , Carcinoma Nasofaríngeo/tratamiento farmacológico , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/tratamiento farmacológico , Adulto , ADN Viral/efectos de los fármacos , Progresión de la Enfermedad , Monitoreo de Drogas/métodos , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/diagnóstico , Infecciones por Virus de Epstein-Barr/virología , Femenino , Fluorodesoxiglucosa F18 , Estudios de Seguimiento , Herpesvirus Humano 4/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Carcinoma Nasofaríngeo/mortalidad , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/mortalidad , Neoplasias Nasofaríngeas/virología , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/virología , Tomografía Computarizada por Tomografía de Emisión de Positrones , Valor Predictivo de las Pruebas , Pronóstico , Criterios de Evaluación de Respuesta en Tumores Sólidos , Factores de Tiempo , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Carga Viral/efectos de los fármacos , Carga Viral/métodosRESUMEN
Combination therapy has been recommended for the treatment of patients harboring multiple drug-resistant hepatitis B virus (HBV). However, we recently demonstrated that monotherapy with tenofovir disoproxil fumarate (TDF) for 48 weeks displayed noninferior efficacy to TDF plus entecavir (ETV) combination therapy in patients with HBV resistant to multiple drugs, including ETV and adefovir. Nonetheless, whether prolonged TDF monotherapy would be safe and increase the virologic response rate in these patients was unclear. Among 192 patients with HBV-resistance mutations to ETV and/or adefovir, who were randomized to receive TDF monotherapy (n = 95) or TDF/ETV combination therapy (n = 97) for 48 weeks, 189 agreed to continue TDF monotherapy (TDF-TDF group) or to switch to TDF monotherapy (TDF/ETV-TDF group) and 180 (93.8%) completed the 144-week study. Serum HBV DNA <15 IU/mL at week 48, the primary efficacy endpoint, was achieved in 66.3% in the TDF-TDF group and 68.0% in the TDF/ETV-TDF group (P = 0.80). At week 144, the proportion with HBV DNA <15 IU/mL increased to 74.5%, which was significantly higher compared with that at week 48 (P = 0.03), without a significant difference between groups (P = 0.46). By on-treatment analysis, a total of 79.4% had HBV DNA <15 IU/mL at week 144. Transient virologic breakthrough occurred in 6 patients, which was due to poor drug adherence. At week 144, 19 patients who had HBV DNA levels >60 IU/mL qualified for genotypic resistance analysis, and 6 retained some of their baseline resistance mutations of HBV. No patients developed additional resistance mutations throughout the study period. CONCLUSION: TDF monotherapy was efficacious and safe for up to 144 weeks, providing an increasing rate of virologic response in heavily pretreated patients with multidrug-resistant HBV. (Hepatology 2017;66:772-783).
Asunto(s)
Antivirales/uso terapéutico , Guanina/análogos & derivados , Hepatitis B Crónica/tratamiento farmacológico , Tenofovir/uso terapéutico , Adulto , Anciano , Análisis de Varianza , ADN Viral/sangre , ADN Viral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Estudios de Seguimiento , Guanina/uso terapéutico , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/patología , Humanos , Masculino , Persona de Mediana Edad , Seguridad del Paciente/estadística & datos numéricos , Ensayos Clínicos Controlados Aleatorios como Asunto , República de Corea , Índice de Severidad de la Enfermedad , Factores de Tiempo , Resultado del Tratamiento , Carga Viral/efectos de los fármacos , Adulto JovenRESUMEN
Regular HIV-1 viral load monitoring is the standard of care to assess antiretroviral therapy effectiveness in resource-rich settings. Persistently elevated viral loads indicate virologic failure (VF), which warrants HIV drug resistance testing (HIVDRT) to allow individualized regimen switches. However, in settings lacking access to HIVDRT, clinical decisions are often made based on symptoms, leading to unnecessary therapy switches and increased costs of care. This work presents a proof-of-concept assay to detect M184V, the most common drug resistance mutation after first-line antiretroviral therapy failure, in a paper format. The first step isothermally amplifies a section of HIV-1 reverse transcriptase containing M184V using a recombinase polymerase amplification (RPA) assay. Then, an oligonucleotide ligation assay (OLA) is used to selectively label the mutant and wild type amplified sequences. Finally, a lateral flow enzyme-linked immunosorbent assay (ELISA) differentiates between OLA-labeled products with or without M184V. Our method shows 100% specificity and 100% sensitivity when tested with samples that contained 200 copies of mutant DNA and 800 copies of wild type DNA prior to amplification. When integrated with sample preparation, this method may detect HIV-1 drug resistance at a low cost and at a rural hospital laboratory.