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The Advisory Committee on Immunization Practices (ACIP) recommended that dengue pre-vaccination screening tests for Dengvaxia administration have at least 98% specificity and 75% sensitivity. This study evaluates the performance of commercial anti-DENV IgG tests to identify tests that could be used for pre-vaccination screening. First, for seven tests, we evaluated sensitivity and specificity in early convalescent dengue virus (DENV) infection, using 44 samples collected 7-30 days after symptom onset and confirmed by RT-PCR. Next, for the five best-performing tests and two additional tests (with and without an external test reader) that became available later, we evaluated performance to detect past dengue infection among a panel of 44 specimens collected in 2018-2019 from healthy 9- to 16-year-old children from Puerto Rico. Finally, a full-scale evaluation was done with the four best-performing tests using 400 specimens from the same population. We used virus focus reduction neutralization test and an in-house DENV IgG ELISA as reference standards. Of seven tests, five showed ≥75% sensitivity in detecting anti-DENV IgG in early convalescent specimens with low cross-reactivity to the Zika virus. For the detection of previous DENV infections, the tests with the highest performance were the Euroimmun NS1 IgG ELISA (sensitivity 84.5%, specificity 97.1%) and CTK Dengue IgG rapid test R0065C with the test reader (sensitivity 76.2% specificity 98.1%). There are IgG tests available that can be used to accurately classify individuals with previous DENV infection as eligible for dengue vaccination to support safe vaccine implementation. IMPORTANCE: The Advisory Committee on Immunization Practices (ACIP) has set forth recommendations that dengue pre-vaccination screening tests must exhibit at least 98% specificity and 75% sensitivity. Our research rigorously assesses the performance of various commercial tests against these benchmarks using well-characterized specimens from Puerto Rico. The findings from our study are particularly relevant given FDA approval and ACIP recommendation of Sanofi Pasteur's Dengvaxia vaccine, highlighting the need for accurate pre-vaccination screening tools.
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Human immunodeficiency virus-type 1 (HIV-1) remains one of the leading contributors to the global burden of disease, and novel antiretroviral agents with alternative mechanisms are needed to cure this infection. Here, we describe an exploratory attempt to optimize the antiretroviral properties of benfluron, a cytostatic agent previously reported to exhibit strong anti-HIV activity likely based on inhibitory actions on virus transcription and Rev-mediated viral RNA export. After obtaining six analogs designed to modify the benzo[c]fluorenone system of the parent molecule, we examined their antiretroviral and toxicity properties together with their capacity to recognize the Rev Recognition Element (RRE) of the virus RNA and inhibit the RRE-Rev interaction. The results indicated that both the benzo[c] and cyclopentanone components of benfluron are required for strong RRE-Rev target engagement and antiretroviral activity and revealed the relative impact of these moieties on RRE affinity, RRE-Rev inhibition, antiviral action and cellular toxicity. These data provide insights into the biological properties of the benzo[c]fluorenone scaffold and contribute to facilitating the design of new anti-HIV agents based on the inhibition of Rev function.
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Fármacos Anti-VIH , Infecciones por VIH , VIH-1 , Humanos , VIH-1/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , ARN Viral/genética , Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , Conformación de Ácido NucleicoRESUMEN
The diagnosis of dengue disease, caused by the dengue virus (DENV) (a flavivirus), often requires serologic testing during acute and early convalescent phases of the disease. Some symptoms of DENV infection, such as nonspecific fever, are similar to those caused by infection with SARS-CoV-2, the virus that causes COVID-19. In studies with few COVID-19 cases, positive DENV immunoglobulin M (IgM) results were reported with various serologic tests, indicating possible cross-reactivity in these tests for DENV and SARS-CoV-2 infections (1,2). DENV antibodies can cross-react with other flaviviruses, including Zika virus. To assess the potential cross-reactivity of SARS-CoV-2, DENV, and Zika virus IgM antibodies, serum specimens from 97 patients from Puerto Rico and 12 U.S.-based patients with confirmed COVID-19 were tested using the DENV Detect IgM Capture enzyme-linked immunosorbent assay (ELISA) (InBios International).* In addition, 122 serum specimens from patients with confirmed dengue and 121 from patients with confirmed Zika virus disease (all from Puerto Rico) were tested using the SARS-CoV-2 pan-Ig Spike Protein ELISA (CDC). Results obtained for DENV, Zika virus IgM, and SARS-CoV-2 antibodies indicated 98% test specificity and minimal levels of cross-reactivity between the two flaviviruses and SARS-CoV-2. These findings indicate that diagnoses of dengue or Zika virus diseases with the serological assays described in this report are not affected by COVID-19, nor do dengue or Zika virus diseases interfere with the diagnosis of COVID-19.
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Anticuerpos Antivirales/sangre , Virus del Dengue/inmunología , Inmunoglobulina M/inmunología , SARS-CoV-2/inmunología , Pruebas Serológicas , Virus Zika/inmunología , COVID-19/diagnóstico , Reacciones Cruzadas/inmunología , Dengue/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Humanos , Puerto Rico , Sensibilidad y Especificidad , Estados Unidos , Infección por el Virus Zika/diagnósticoRESUMEN
The aim of the present study is to report the isolation, structural elucidation, and antiviral evaluation of four new withanolide-type steroids, named nicansteroidins A-D (1-4), together with nine related known compounds (5-13) isolated from the aerial parts of Physalis nicandroides. Their structures were established based on an extensive spectroscopic analysis, including 1D and 2D NMR techniques. Outstandingly, nicansteroidins A and B possess an unusual side chain with an exocyclic double bond on the δ-lactone system, whereas nicansteroidins C and D have an uncommon cycloperoxide functionality in ring A as distinct structural motifs. Their biological evaluation as inhibitors of human immunodeficiency virus type 1 replication revealed that two compounds from this series, 7 and 13, displayed strong inhibition of HIV-1 replication with IC50 values lower than 2 µM. Moreover, cellular mechanism experiments showed that the main target of these compounds in the HIV replication cycle is viral transcription. This study is the first report of withanolide-type steroids as HIV inhibitors and provides insight into their potential as candidates for further preclinical studies.
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Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Physalis/química , Replicación Viral/efectos de los fármacos , Witanólidos/farmacología , Línea Celular , El Salvador , VIH-1/fisiología , Humanos , Estructura Molecular , Componentes Aéreos de las Plantas/químicaRESUMEN
Cross-reactivity within flavivirus antibody assays, produced by shared epitopes in the envelope proteins, can complicate the serological diagnosis of Zika virus (ZIKAV) infection. We assessed the utility of the plaque reduction neutralization test (PRNT) to confirm recent ZIKAV infections and rule out misleading positive immunoglobulin M (IgM) results in areas with various levels of past dengue virus (DENV) infection incidence. We reviewed PRNT results of sera collected for diagnosis of ZIKAV infection from 1 January through 31 August 2016 with positive ZIKAV IgM results, and ZIKAV and DENV PRNTs were performed. PRNT result interpretations included ZIKAV, unspecified flavivirus, DENV infection, or negative. For this analysis, ZIKAV IgM was considered false positive for samples interpreted as a DENV infection or negative. In U.S. states, 208 (27%) of 759 IgM-positive results were confirmed to be ZIKAV compared to 11 (21%) of 52 in the U.S. Virgin Islands (USVI), 15 (15%) of 103 in American Samoa, and 13 (11%) of 123 in Puerto Rico. In American Samoa and Puerto Rico, more than 80% of IgM-positive results were unspecified flavivirus infections. The false-positivity rate was 27% in U.S. states, 18% in the USVI, 2% in American Samoa, and 6% in Puerto Rico. In U.S. states, the PRNT provided a virus-specific diagnosis or ruled out infection in the majority of IgM-positive samples. Almost a third of ZIKAV IgM-positive results were not confirmed; therefore, providers and patients must understand that IgM results are preliminary. In territories with historically higher rates of DENV transmission, the PRNT usually could not differentiate between ZIKAV and DENV infections.
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Anticuerpos Antivirales/sangre , Virus del Dengue/inmunología , Dengue/epidemiología , Inmunoglobulina M/sangre , Infección por el Virus Zika/diagnóstico , Virus Zika/inmunología , Samoa Americana/epidemiología , Reacciones Cruzadas , Reacciones Falso Positivas , Femenino , Flavivirus/inmunología , Humanos , Incidencia , Masculino , Pruebas de Neutralización , Puerto Rico/epidemiología , Estados Unidos/epidemiología , Islas Virgenes de los Estados Unidos/epidemiología , Infección por el Virus Zika/epidemiología , Infección por el Virus Zika/virologíaRESUMEN
BACKGROUND: Anti-dengue virus (DENV) immunoglobulin M (IgM) seroconversion has been the reference standard for dengue diagnosis. However, paired specimens are rarely obtained, and the interval for this testing negates its usefulness in guiding clinical case management. The presence of DENV viremia and appearance of IgM during the febrile phase of dengue provides the framework for dengue laboratory diagnosis by using a single serum specimen. METHODS: Archived paired serum specimens (n = 1234) from patients with laboratory-confirmed dengue from 2005 through 2011 were used to determine the diagnostic performance of real-time reverse transcription polymerase chain reaction (RT-PCR), for detection of DENV serotypes 1-4, and enzyme-linked immunosorbent assays (ELISAs), for detection of DENV nonstructural protein 1 (NS1) antigen and anti-DENV IgM. RESULTS: During 1-3 days after illness onset, real-time RT-PCR and NS1 antigen testing detected 82%-69% and 90%-84% of cases, respectively, as viremia levels declined, while anti-DENV IgM ELISA detected 5%-41% of cases as antibody appeared. Over the 10-day period of the febrile phase of dengue, the cumulative effect of using these 3 types of tests in a diagnostic algorithm confirmed ≥90% of dengue cases. CONCLUSIONS: The use of molecular or NS1 antigen tests to detect DENV and one to detect anti-DENV IgM in a single serum specimen collected during the first 10 days of illness accurately identified ≥90% of dengue primary and secondary cases.
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Anticuerpos Antivirales/sangre , Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Pruebas Diagnósticas de Rutina/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina M/sangre , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , ARN Viral/sangre , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Adulto JovenRESUMEN
As a part of our investigation into new anti-HIV agents, we report herein the isolation, structure elucidation, and biological activity of six new (1-6) and 20 known (7-26) pentacyclic lupane-type triterpenoids from the stem of Cassine xylocarpa and root bark of Maytenus cuzcoina. Their stereostructures were elucidated on the basis of spectroscopic and spectrometric methods, including 1D and 2D NMR techniques. To gain a more complete understanding of the structural requirements for anti-HIV activity, derivatives 27-48 were prepared by chemical modification of the main secondary metabolites. Sixteen compounds from this series displayed inhibitory effects of human immunodeficiency virus type 1 replication with IC50 values in the micromolar range, highlighting compounds 12, 38, and 42 (IC50 4.08, 4.18, and 1.70 µM, respectively) as the most promising anti-HIV agents.
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Fármacos Anti-VIH/aislamiento & purificación , Fármacos Anti-VIH/farmacología , Celastraceae/química , Maytenus/química , Triterpenos/aislamiento & purificación , Triterpenos/farmacología , Fármacos Anti-VIH/química , Antineoplásicos Fitogénicos/química , VIH-1/efectos de los fármacos , Humanos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Perú , Corteza de la Planta/química , Tallos de la Planta/química , Relación Estructura-Actividad , Triterpenos/químicaRESUMEN
ETHNOPHARMACOLOGY RELEVANCE: Lepidium meyenii Walp. (maca) has been traditionally used for centuries in the Central Andes region both as food and as medicine. In the last decades, its fertility enhancer properties have gained importance, with the majority of the scientific literature related to this topic. However, other traditional uses are less known as metabolic or infectious diseases. AIM OF THE STUDY: The main purpose of this study is to investigate the anti-infectious activity of L. meyenii, specifically in HIV-1 infection. There are previous reports of the transcriptional related activity of L. meyenii extracts in human T lymphocytes via transcription factors as NF-κB. Since T lymphocytes are the main target of HIV-1 infection and NF-κB is strongly involved in HIV-1 transcription, L. meyenii could display antiviral activity. MATERIAL AND METHODS: Chromatography and spectroscopy techniques were used to isolate and identify the compounds in the active extracts. An antiviral assay system based on recombinant viruses was used to evaluate the anti-HIV activity. Cell toxicity was tested for all the extracts and compounds. Viral entry was studied using VSV-HIV chimera viruses and reverse transcription and viral integration were studied by qPCR of viral DNA in infected cells. Finally, viral transcription was studied in primary lymphocytes transfected with HIV-1 or NF-κB luciferase reporter plasmids. RESULTS: n-Hexane extracts of purple maca displayed anti-HIV activity in an in vitro assay. A bioassay-guided fractionation led to the identification of three thiadiazole alkaloids with antiviral activity. All the compounds were able to inhibit HIV infection of MT-2 cell lines and primary lymphocytes (PBMCs) with IC50 values in the low micromolar range. The mechanism of action differs between the three compounds: one of them showed activity on viral entry, and all the three compounds inhibited viral integration at low concentrations. Remarkably, none of the compounds inhibited reverse transcription or viral transcription. CONCLUSIONS: n-Hexane extracts of the purple ecotype of L. meyenii inhibit HIV-1 infection in vitro and three active thiadiazole alkaloids were isolated acting mainly on viral integration and viral entry.
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The Advisory Committee on Immunization Practices (ACIP) recommended that dengue pre-vaccination screening tests for Dengvaxia administration have at least 98% specificity and 75% sensitivity. This study evaluates the performance of commercial anti-DENV IgG tests to identify tests that could be used for pre-vaccination screening. First, for 7 tests, we evaluated sensitivity and specificity in early convalescent dengue virus (DENV) infection, using 44 samples collected 7-30 days after symptom onset and confirmed by RT-PCR. Next, for the 5 best performing tests and two additional tests (with and without an external test reader) that became available later, we evaluated performance to detect past dengue infection among a panel of 44 specimens collected in 2018-2019 from healthy 9-16-year-old children from Puerto Rico. Finally, a full-scale evaluation was done with the 4 best performing tests using 400 specimens from the same population. We used virus focus reduction neutralization test and an in-house DENV IgG ELISA as reference standards. Of seven tests, five showed ≥75% sensitivity detecting anti-DENV IgG in early convalescent specimens with low cross-reactivity to Zika virus. For the detection of previous DENV infections the tests with the highest performance were the Euroimmun NS1 IgG ELISA (sensitivity 84.5%, specificity 97.1%) and CTK Dengue IgG rapid test R0065C with the test reader (sensitivity 76.2% specificity 98.1%). There are IgG tests available that can be used to accurately classify individuals with previous DENV infection as eligible for dengue vaccination to support safe vaccine implementation.
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The complex between the Rev protein of HIV-1 and the Rev Recognition Element (RRE) within the virus RNA promotes nuclear export of unspliced or incompletely spliced viral transcripts and is required for virus transmission. Here, we have screened a virtual collection of compounds using a pharmacophore based on the chemical similarity of previously characterized inhibitors to identify new chemical scaffolds blocking the RRE-Rev interaction. The best molecules discovered with this strategy inhibited the complex by binding to the RRE and exhibited substantial antiretroviral activity (between 0.582 and 11.3 µM EC50 values) likely associated to inhibitory actions on viral transcription and Rev function. These results have allowed us to identify structural features required for RRE-Rev inhibition as well as to add new compounds to the pool of possible candidates for developing antiretroviral agents based on blockage of HIV-1 RNA biogenesis.
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VIH-1 , VIH-1/fisiología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Farmacóforo , ARN Viral/metabolismo , Transporte Activo de Núcleo CelularRESUMEN
Broadly neutralizing antibodies (bnAbs) bind and neutralize diverse HIV isolates and demonstrate protective effects in primate models and humans against specific isolates. To develop an effective HIV vaccine, it is widely believed that inducing these antibodies is crucial. However, the high somatic hypermutation in bnAbs and the limited affinity of HIV Env proteins for bnAb germline precursors suggest that extended antigen exposure is necessary for their production. Consequently, HIV vaccine research is exploring complex sequential vaccination strategies to guide the immune response through maturation stages. In this context, the exploration of the factors linked to the generation of these antibodies across diverse age groups becomes critical. In this study, we assessed the anti-HIV-1 neutralization potency and breadth in 108 aviremic adults and 109 aviremic children under 15 years of age who were receiving ART. We used a previously described minipanel of recombinant viruses and investigated the factors associated with neutralization in these individuals. We identified individuals in both groups who were capable of neutralizing viruses from three different subtypes, with greater cross-neutralization observed in the adult group (49.0% vs. 9.2%). In both groups, we observed an inverse association between neutralization breadth and the CD4+/CD8+ ratio, as well as a direct association with the time to ART initiation. However, we found no association with time post-infection, cumulative ART duration, or CD8+ cell levels. The present study demonstrates that children receiving antiretroviral therapy generate broadly neutralizing responses to HIV-1, albeit with lower magnitude compared to adults. We also observed that neutralization breadth is associated with CD4+/CD8+ levels and time to treatment initiation in both children and adults living with HIV-1. Our interpretation of these results is that a delay in ART initiation could have prolonged the antigenic stimulation associated with viral replication and thus facilitate the capacity to elicit long-lasting broadly neutralizing responses. These results corroborate prior findings that show that HIV-1-neutralizing responses can persist for years, even at low antigen levels, implying an HIV-1 vaccine may induce lasting neutralizing antibody response.
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Most of the studies using the colorectal tissue explants challenge model have been conducted after one single dose and before reaching a steady state. We consider that longer exposure as in 28-day postexposure prophylaxis (PEP) course and in an at-risk setting, such as after a sexual risk exposure to HIV could give us valuable information about these drugs. In a substudy we assessed pharmacokinetics, changes on immune system and ex-vivo rectal mucosal susceptibility to HIV-1 infection after taking maraviroc (MVC), raltegravir (RAL), and ritonavir-boosted lopinavir (LPV/r) PEP-based regimens in 30 men who have sex with men. Participants received 28 days of twice-daily MVC (n = 11), RAL (n = 10) or LPV/r (n = 9) all with tenofovir/emtricitabine (TDF/FTC) backbone. Blood, rectal fluid, and rectal tissue samples were collected at days 7, 28, and 90 after starting PEP. The samples obtained at day 90 were considered baseline. All studied antiretrovirals were quantifiable at 7 and 28 days in all tissues. Activation markers were increased in CD4 mucosal mononuclear cells (MMCs) after 28 days of MVC: CD38 + 68.5 versus 85.1, p = .008 and CD38+DR +16.1 versus 26.7, p = .008. Exposure to MVC at both endpoints (7 and 28 days) was associated with significant suppression of HIV-1BAL (p = .005 and p = .028), but we did not observe this effect with RAL or LPV/r. Merging together changes in MMC in all arms, we found a positive correlation in the CD8 T cell lineage between the infectivity at day 7 and activation (CD38+ r = 0.43, p = .025, DR + r = 0.547, p = .003 and 38+DR+ r = 0.526, p = .05), senescence (CD57+CD28- r = 0.479, p = .012), naive cells (RA+CCR7+ r = 0.484, p = .01), and CCR5 expression (r = 0.593, p = .001). We conclude that MVC in combination with TDF/FTC was associated with viral suppression in rectal explants and that overall ex-vivo HIV infectivity correlated with activation and senescence in CD8 MMCs.
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Fármacos Anti-VIH , Infecciones por VIH , Minorías Sexuales y de Género , Masculino , Humanos , Maraviroc , Raltegravir Potásico/uso terapéutico , Lopinavir/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Fármacos Anti-VIH/uso terapéutico , Homosexualidad Masculina , Emtricitabina/uso terapéutico , Ritonavir/uso terapéutico , Profilaxis PosexposiciónRESUMEN
Murine models of Mycobacterium tuberculosis infection are essential tools in drug discovery. Here we describe a fast standardized 9-day acute assay intended to measure the efficacy of drugs against M. tuberculosis growing in the lungs of immunocompetent mice. This assay is highly reproducible, allows good throughput, and was validated for drug lead optimization using isoniazid, rifampin, ethambutol, pyrazinamide, linezolid, and moxifloxacin.
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Antituberculosos/farmacología , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Ratones Endogámicos C57BL , Tuberculosis Pulmonar/tratamiento farmacológico , Acetamidas/farmacología , Animales , Compuestos Aza/farmacología , Etambutol/farmacología , Fluoroquinolonas , Inmunocompetencia , Exposición por Inhalación , Isoniazida/farmacología , Linezolid , Ratones , Moxifloxacino , Oxazolidinonas/farmacología , Pirazinamida/farmacología , Quinolinas/farmacología , Reproducibilidad de los Resultados , Rifampin/farmacología , Tuberculosis Pulmonar/inmunologíaRESUMEN
Small synthetic molecules mimicking the three-dimensional structure of α-helices may find applications as inhibitors of therapeutically relevant protein-protein and protein-nucleic acid interactions. However, the design and use of multi-facial helix mimetics remains in its infancy. Here we describe the synthesis and application of novel bilaterally substituted p-terphenyl compounds containing positively-charged aminoalkyl groups in relative 1,4 positions across the aromatic scaffold. These compounds were specifically designed to mimic all faces of the arginine-rich α-helix of the HIV-1 protein Rev, which forms deeply embedded RNA complexes and plays key roles in the virus replication cycle. Two of these molecules recognized the Rev site in the viral RNA and inhibited the formation of the RRE-Rev ribonucleoprotein complex, a currently unexploited target in HIV chemotherapy. Cellular assays revealed that the most active compounds blocked HIV-1 replication with little toxicity, and likely exerted this effect through a multi-target mechanism involving inhibition of viral LTR promoter-dependent transcription and Rev function. Further development of this scaffold may open new avenues for targeting nucleic acids and may complement current HIV therapies, none of which involve inhibitors interfering with the gene regulation processes of the virus.
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Fármacos Anti-VIH/química , VIH-1/química , Conformación de Ácido Nucleico , ARN Viral/química , Compuestos de Terfenilo/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Línea Celular , Humanos , Conformación Proteica en Hélice alfaAsunto(s)
Fármacos Anti-VIH/farmacología , Diseño de Fármacos , ARN/química , Compuestos de Terfenilo/farmacología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Sitios de Unión , Relación Dosis-Respuesta a Droga , VIH-1/efectos de los fármacos , Humanos , Estructura Molecular , Relación Estructura-Actividad , Compuestos de Terfenilo/síntesis química , Compuestos de Terfenilo/química , Replicación Viral/efectos de los fármacos , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The Rev protein of HIV-1 binds to the Rev Recognition Element (RRE) in the virus RNA to promote nuclear export of unspliced and partially spliced transcripts, an essential step in the virus transmission cycle. Here, we describe the screening of a library of chemically diverse compounds with an assay based on monitoring the interaction between the RNA-binding α-helix of Rev and its high-affinity binding site in the RRE. This screen allowed the identification of a benzofluorenone compound that inhibited the formation of the full-length RRE-Rev ribonucleoprotein by associating to the RRE, and blocked HIV-1 transcription and Rev action in cells. This molecule, previously studied as a cytostatic agent, had substantial antiretroviral activity. Together with other screening hits, it provides a new chemical scaffold for the development of antiretroviral agents based on blockage of HIV-1 RNA biogenesis.
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Antivirales/farmacología , VIH-1/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Antivirales/química , Línea Celular , Evaluación Preclínica de Medicamentos , Humanos , Estructura Molecular , ARN Viral/metabolismo , Elementos de Respuesta , Relación Estructura-ActividadRESUMEN
Current research on antiretroviral therapy is mainly focused in the development of new formulations or combinations of drugs belonging to already known targets. However, HIV-1 infection is not cured by current therapy and thus, new approaches are needed. Bevirimat was developed by chemical modification of betulinic acid, a lupane-type pentacyclic triterpenoid (LPT), as a first-in-class HIV-1 maturation inhibitor. However, in clinical trials, bevirimat showed less activity than expected because of the presence of a natural mutation in Gag protein that conferred resistance to a high proportion of HIV-1 strains. In this work, three HIV-1 inhibitors selected from a set of previously screened LPTs were investigated for their targets in the HIV-1 replication cycle, including their maturation inhibitor effect. LPTs were found to inhibit HIV-1 infection acting as promiscuous compounds with several targets in the HIV-1 replication cycle. LPT12 inhibited HIV-1 infection mainly through reverse transcription, integration, viral transcription, viral proteins (Gag) production and maturation inhibition. LPT38 did it through integration, viral transcription or Gag production inhibition and finally, LPT42 inhibited reverse transcription, viral transcription or Gag production. The three LPTs inhibited HIV-1 infection of human primary lymphocytes and infections with protease inhibitors and bevirimat resistant HIV-1 variants with similar values of IC50. Therefore, we show that the LPTs tested inhibited HIV-1 infection through acting on different targets depending on their chemical structure and the activities of the different LPTs vary with slight structural alterations. For example, of the three LPTs under study, we found that only LPT12 inhibited infectivity of newly-formed viral particles, suggesting a direct action on the maturation process. Thus, the multi-target behavior gives a potential advantage to these compounds since HIV-1 resistance can be overcome by modulating more than one target.
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New antiretroviral agents with alternative mechanisms are needed to complement the combination therapies used to treat HIV-1 infections. Here we report the identification of bioavailable molecules that interfere with the gene expression processes of HIV-1. The compounds were detected by screening a small library of FDA-approved drugs with an assay based on measuring the displacement of Rev, and essential virus-encoded protein, from its high-affinity RNA binding site. The antiretroviral activity of two hits was based on interference with post-integration steps of the HIV-1 cycle. Both hits inhibited RRE-Rev complex formation in vitro, and blocked LTR-dependent gene expression and viral transcription in cellular assays. The best compound altered the splicing pattern of HIV-1 transcripts in a manner consistent with Rev inhibition. This mechanism of action is different from those used by current antiretroviral agents. The screening hits recognized the Rev binding site in the viral RNA, and the best compound did so with substantial selectivity, allowing the identification of a new RNA-binding scaffold. These results may be used for developing novel antiretroviral drugs.
Asunto(s)
Fármacos Anti-VIH/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , VIH-1/efectos de los fármacos , ARN Viral/metabolismo , Elementos de Respuesta/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Fármacos Anti-VIH/efectos adversos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Clomifeno/efectos adversos , Clomifeno/farmacología , Ciproheptadina/efectos adversos , Ciproheptadina/farmacología , Evaluación Preclínica de Medicamentos , Genes Reporteros/efectos de los fármacos , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Empalme del ARN/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
OBJECTIVE: To investigate the toxicity and activity against HIV of 5-hydroxytyrosol as a potential microbicide. DESIGN: The anti-HIV-1 activity of 5-hydroxytyrosol, a polyphenolic compound, was tested against wild-type HIV-1 and viral clones resistant to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and integrase inhibitors. In addition to its activity against founder viruses, different viral subtypes and potential synergy with tenofovir disoproxil fumarate, lamivudine and emtricitabine was also tested. 5-Hydroxytyrosol toxicity was evaluated in vivo in rabbit vaginal mucosa. METHODS: We have cloned pol gene from drug-resistant HIV-1 isolated from infected patients and env gene from Fiebeg III/IV patients or A, C, D, E, F and G subtypes in the NL4.3-Ren backbone. 5-Hydroxytyrosol anti-HIV-1 activity was evaluated in infections of MT-2, U87-CCR5 or peripheral blood mononuclear cells preactivated with phytohemagglutininâ+âinterleukin-2 with viruses obtained through 293T transfections. Inhibitory concentration 50% and cytotoxic concentration 50% were calculated. Synergy was analysed according to Chou and Talalay method. In-vivo toxicity was evaluated for 14 days in rabbit vaginal mucosa. RESULTS: 5-Hydroxytyrosol inhibited HIV-1 infections of recombinant or wild-type viruses in all the target cells tested. Moreover, 5-hydroxytyrosol showed similar inhibitory concentration 50% values for infections with NRTIs, NNRTIs, protease inhibitors and INIs resistant viruses; founder viruses and all the subtypes tested. Combination of 5-hydroxytyrosol with tenofovir was found to be synergistic, whereas it was additive with lamivudine and emtricitabine. In-vivo toxicity of 5-hydroxytyrosol was very low even at the highest tested doses. CONCLUSION: 5-Hydroxytyrosol displayed a broad anti-HIV-1 activity in different cells systems in the absent of in-vivo toxicity, therefore supporting its candidacy as a potential new class of microbicides.
Asunto(s)
Antiinfecciosos/farmacología , VIH-1/efectos de los fármacos , Alcohol Feniletílico/análogos & derivados , Animales , Antiinfecciosos/administración & dosificación , Antiinfecciosos/efectos adversos , Células Cultivadas , Sinergismo Farmacológico , Femenino , Actividades Humanas , Humanos , Leucocitos Mononucleares/virología , Pruebas de Sensibilidad Microbiana , Alcohol Feniletílico/administración & dosificación , Alcohol Feniletílico/efectos adversos , Alcohol Feniletílico/farmacología , ConejosRESUMEN
Dengue is a potentially fatal acute febrile illness caused by the mosquito-borne dengue viruses (DENV-1 to -4). To estimate DENV seroincidence in school-aged children, a 1-year prospective cohort study was conducted in Patillas, Puerto Rico; 10- to 18-year-olds (N = 345) were randomly selected from 13 public schools. At enrollment, 49.8% of the entire cohort had DENV immunoglobulin G (IgG) anti-DENV antibodies, and there were individuals with neutralizing antibodies specific to each of the four DENV. The mean age of participants with incident DENV infection was 13.4 years. The 1-year seroincidence rate was 5.6%, and 61.1% of infections were inapparent. Having IgG anti-DENV at enrollment was associated with seroincidence (risk ratio = 6.8). Acute febrile illnesses during the study period were captured by a fever diary and an enhanced and passive surveillance system in the municipios of Patillas and Guayama. In summary, at enrollment, nearly one-half of the participants had a prior DENV infection, with the highest incidence in the 10- to 11-year-olds, of which most were inapparent infections, and symptomatic infections were considered mild.