Pharmacologic control of homeostatic and antigen-driven proliferation to target HIV-1 persistence.

The presence of latent human immunodeficiency virus 1 (HIV-1) in quiescent memory CD4 + T cells represents a major barrier to viral eradication. Proliferation of memory CD4 + T cells is the primary mechanism that leads to persistence of the latent reservoir, despite effective antiretroviral therapy (ART). Memory CD4 + T cells are long-lived and can proliferate through two mechanisms: homeostatic proliferation via γc-cytokine stimulation or antigen-driven proliferation. Therefore, therapeutic modalities that perturb homeostatic and antigen-driven proliferation, combined with ART, represent promising strategies to reduce the latent reservoir. In this study, we investigated a library of FDA-approved oncology drugs to determine their ability to inhibit homeostatic and/or antigen-driven proliferation. We confirmed potential hits by evaluating their effects on proliferation in memory CD4 + T cells from people living with HIV-1 on ART (PLWH) and interrogated downstream signaling of γc-cytokine stimulation. We found that dasatinib and ponatinib, tyrosine kinase inhibitors, and trametinib, a MEK inhibitor, reduced both homeostatic and antigen-driven proliferationby >65%, with a reduction in viability <45%, ex vivo. In memory CD4 + T cells from PLWH, only dasatinib restricted both homeostatic and antigen-driven proliferation and prevented spontaneous rebound, consistent with promoting a smaller reservoir size. We show that dasatinib restricts IL-7 induced proliferation through STAT5 phosphorylation inhibition. Our results establish that the anti-cancer agent dasatinib is an exciting candidate to be used as an anti-proliferative drug in a clinical trial, since it efficiently blocks proliferation and iswell tolerated in patients with chronic myeloid leukemia (CML).

Ethanolic extract of Artemisia campestris subsp. glutinosa (Besser) Batt. inhibits HIV-1 replication in vitro through the activity of terpenes and flavonoids on viral entry and NF-κB pathway.

ETHNO-PHARMACOLOGICAL RELEVANCE: The genus Artemisia spp. is well known for its anti-infectious properties and its high content in anti-infectious compounds, like the well-known sweet wormwood (Artemisia annua L.). Another Artemisia species, Artemisia campestris subsp. glutinosa (Besser) Batt., field wormwood, has been traditionally used as medicinal plant in the Mediterranean region. AIM OF THE STUDY: The aim of this study is to investigate the anti-HIV activity of field wormwood, to identify the compounds responsible for this activity and their structure and mechanism of action. MATERIALS AND METHODS: Antiviral activity of isolated compounds and extracts was evaluated in HIV-1 infections of lymphoblastoid cells. We also evaluated the mechanism of action of isolated compounds. Viral entry was studied comparing the inhibitory effect of isolated compounds on wild type HIV-1 and VSV pseudotyped HIV-1. To assess the viral transcriptional effect, plasmids encoding luciferase reporter genes under the control of the whole genome of HIV-1 or NF-κB or Sp1 transcription factors were transfected in the presence of the compounds under evaluation. Finally, antioxidant activity was assessed by quantitation of reduced and total glutathione in treated cell cultures. RESULTS: Ethanolic and aqueous extracts of Artemisia campestris subsp. glutinosa (Besser) Batt. subsp. glutinosa displayed anti-HIV activity in vitro, although ethanolic extract was more powerful (IC50 14.62 µg/mL). Bio-guided ethanolic extract fractionation leads to the isolation and characterization of two terpenes, damsin and canrenone, and four flavonoids, 6, 2', 4'-trimethoxyflavone, acerosin, cardamonin and xanthomicrol. All the isolated compounds inhibited HIV-1 replication in vitro with IC50 values between the middle nanomolar and the low micromolar range. Their anti-HIV mechanism of action is due to the bloking of viral entry and/or transcription inhibition, without correlation with the antioxidant activity, through interference with the cellular transcription factors NF-κB and Sp1, which are targets that are not currently reached by antiretroviral therapy. CONCLUSION: We describe here the anti-HIV activity of field wormwood, Artemisia campestris subsp. glutinosa (Besser) Batt., and the isolation and study of the mechanism of action of two terpenes and four flavonoids, responsible, at least in part, for its activity, through the inhibition of two different cellular targets affecting the HIV replication cycle. The activity of these compounds in cellular targets could explain why plant extracts can be used in the treatment of different diseases. Besides, the presence of several compounds with dual and different mechanisms of action could prove useful in the treatment of HIV-1 infection, since it could aid to overcome drug resistances and simplify drug therapy. This work is a further step in understanding the anti-infectious activity of wormwood species and their use in treating infectious diseases.

4-Deoxyphorbol inhibits HIV-1 infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat.

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy proposes to eliminate these viral reservoirs by pharmacologically reactivating the latently infected T cells. We show here that a 4-deoxyphorbol ester derivative isolated from Euphorbia amygdaloides ssp. semiperfoliata, 4ß-dPE A, reactivates HIV-1 from latency and could potentially contribute to decrease the viral reservoir. 4ß-dPE A shows two effects in the HIV replication cycle, infection inhibition and HIV transactivation, similarly to other phorboids PKC agonists such PMA and prostratin and to other diterpene esters such SJ23B. Our data suggest 4ß-dPE A is non-tumorigenic, unlike the related compound PMA. As the compounds are highly similar, the lack of tumorigenicity by 4ß-dPE A could be due to the lack of a long side lipophilic chain that is present in PMA. 4ß-dPE activates HIV transcription at nanomolar concentrations, lower than the concentration needed by other latency reversing agents (LRAs) such as prostratin and similar to bryostatin. PKCθ/MEK activation is required for the transcriptional activity, and thus, anti-latency activity of 4ß-dPE A. However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be independent of PCK/MEK, suggesting the existence of at least two different targets for 4ß-dPE A. Furthermore, NF-κb transcription factor is involved in 4ß-dPE HIV reactivation, as previously shown for other PKCs agonists. We also studied the effects of 4ß-dPE A in combination with other LRAs. When 4ß-dPE A was combined with another PKC agonists such as prostratin an antagonic effect was achieved, while, when combined with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. Interestingly, the latency reversing effect of the combination was synergistically diminishing the EC50 value but also increasing the efficacy showed by the drugs alone. In addition, combinations of 4ß-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would not interefer with antiretroviral therapy (ART). Finally, 4ß-dPE A induced latent HIV reactivation in CD4 + T cells of infected patients under ART at similar levels than the tumorigenic phorbol derivative PMA, showing a clear reactivation effect. In summary, we describe here the mechanism of action of a new potent deoxyphorbol derivative as a latency reversing agent candidate to decrease the size of HIV reservoirs.

Association of a single nucleotide polymorphism in the ubxn6 gene with long-term non-progression phenotype in HIV-positive individuals.

OBJECTIVES: The long-term non-progressors (LTNPs) are a heterogeneous group of HIV-positive individuals characterized by their ability to maintain high CD4+ T-cell counts and partially control viral replication for years in the absence of antiretroviral therapy. The present study aims to identify host single nucleotide polymorphisms (SNPs) associated with non-progression in a cohort of 352 individuals. METHODS: DNA microarrays and exome sequencing were used for genotyping about 240 000 functional polymorphisms throughout more than 20 000 human genes. The allele frequencies of 85 LTNPs were compared with a control population. SNPs associated with LTNPs were confirmed in a population of typical progressors. Functional analyses in the affected gene were carried out through knockdown experiments in HeLa-P4, macrophages and dendritic cells. RESULTS: Several SNPs located within the major histocompatibility complex region previously related to LTNPs were confirmed in this new cohort. The SNP rs1127888 (UBXN6) surpassed the statistical significance of these markers after Bonferroni correction (q = 2.11 × 10-6). An uncommon allelic frequency of rs1127888 among LTNPs was confirmed by comparison with typical progressors and other publicly available populations. UBXN6 knockdown experiments caused an increase in CAV1 expression and its accumulation in the plasma membrane. In vitro infection of different cell types with HIV-1 replication-competent recombinant viruses caused a reduction of the viral replication capacity compared with their corresponding wild-type cells expressing UBXN6. CONCLUSIONS: A higher prevalence of Ala31Thr in UBXN6 was found among LTNPs within its N-terminal region, which is crucial for UBXN6/VCP protein complex formation. UBXN6 knockdown affected CAV1 turnover and HIV-1 replication capacity.

HLA-B*57 and IFNL4-related polymorphisms are associated with protection against HIV-1 disease progression in controllers.

Background: HIV-1-controllers maintain HIV-1 viremia at low levels (normally <2000 HIV-RNA copies/mL) without antiretroviral treatment. However, some HIV-1-controllers have evidence of immunologic progression with marked CD4+T-cell decline. We investigated host genetic factors associated with protection against CD4+T-cell loss in HIV-1-controllers. Methods: We analysed the association of interferon lambda 4 (IFNL4)-related polymorphisms and HLA-B haplotypes within Long Term Non-Progressor HIV-1-controllers ((LTNP-C), defined by maintaining CD4+T-cells counts >500 cells/mm3 for more than 7 years after HIV-1 diagnosis) versus non-LTNP-C, who developed CD4+T-cells counts <500 cells/mm3 Both a Spanish study cohort (n=140) and an international validation cohort (n=914) were examined. Additionally, in a subgroup of individuals HIV-1-specific T-cell responses and soluble cytokines were analysed RESULTS: HLA-B*57 was independently associated with the LTNP-C phenotype (OR=3.056 (1.029-9.069) p=0.044 and OR=1.924 (1.252-2.957) p=0.003) while IFNL4 genotypes represented independent factors for becoming non-LTNP-C (TT/TT, ss469415590, OR=0.401 (0.171-0.942) p=0.036 or A/A, rs12980275, OR=0.637 (0.434-0.934) p=0.021) in the Spanish and validation cohort, respectively, after adjusting for sex, age at HIV-1 diagnosis, IFNL4-related polymorphisms and different HLA-B haplotypes. LTNP-C showed lower plasma IP-10 (p=0.019) and higher IFN-γ (p=0.02) levels than the HIV-1-controllers with diminished CD4+T-cell numbers. Moreover, LTNP-C exhibited higher quantities of IL2+CD57- and IFN-γ+CD57- HIV-1-specific CD8+T-cells (p=0.002 and 0.041, respectively) than non-LTNP-C. Conclusions: We have defined genetic markers able to segregate stable HIV-1-controllers from those who experience CD4+T-cell decline. These findings allow for identification of HIV-1-controllers at risk for immunologic progression, and provide avenues for personalized therapeutic interventions and precision medicine for optimizing clinical care of these individuals.

Detection of Broadly Neutralizing Activity within the First Months of HIV-1 Infection.

UNLABELLED: A fraction of HIV-1 patients are able to generate broadly neutralizing antibodies (bNAbs) after 2 to 4 years of infection. In rare occasions such antibodies are observed close to the first year of HIV-1 infection but never within the first 6 months. In this study, we analyzed the neutralization breadth of sera from 157 antiretroviral-naive individuals who were infected for less than 1 year. A range of neutralizing activities was observed with a previously described panel of six recombinant viruses from five different subtypes (M. Medina-Ramirez et al., J Virol 85:5804-5813, 2011, http://dx.doi.org/10.1128/JVI.02482-10). Some sera were broadly reactive, predominantly targeting envelope epitopes within the V2 glycan-dependent region. The neutralization breadth was positively associated with time postinfection (P = 0.0001), but contrary to what has been reported for chronic infections, no association with the viral load was observed. Notably, five individuals within the first 6 months of infection (two as early as 77 and 96 days postinfection) showed substantial cross-neutralization. This was confirmed with an extended panel of 20 Env pseudoviruses from four different subtypes (two in tier 3, 14 in tier 2, and four in tier 1). Sera from these individuals were capable of neutralizing viruses from four different subtypes with a geometric mean 50% infective dose (ID50) between 100 and 800. These results indicate that induction of cross-neutralizing responses, albeit rare, is achievable even within 6 months of HIV-1 infection. These observations encourage the search for immunogens able to elicit this kind of response in preventive HIV-1 vaccine approaches. IMPORTANCE: There are very few individuals able to mount broadly neutralizing activity (bNA) close to the first year postinfection. It is not known how early in the infection cross-neutralizing responses can be induced. In the present study, we show that bNAbs, despite being rare, can be induced much earlier than previously thought. The identification of HIV-1-infected patients with these activities within the first months of infection and characterization of these responses will help in defining new immunogen designs and neutralization targets for vaccine-mediated induction of bNAbs.

Estudio prospectivo observacional para evaluar, desde la perspectiva del paciente, el consejo sanitario preoperatorio administrado en fumadores intervenidos en Cirugía Mayor Ambulatoria / Prospective observational study to evaluate from the patient’s perspective the anesthesiologist-delivered interventions to quit smoking before ambulatory surgery

Introducción: Los pacientes fumadores presentan un riesgo aumentado de complicaciones perioperatorias, incluyendo los pacientes intervenidos en cirugía ambulatoria siendo las más relevantes las complicaciones respiratorias y las de infección de la herida quirúrgica. La consulta de anestesia representa una oportunidad excelente para promover el cese del tabaquismo. Encuestas realizadas en diferentes países muestran que los anestesiólogos administran consejo sanitario de forma irregular a los pacientes fumadores que precisan una intervención quirúrgica. Sin embargo, ningún estudio ha evaluado directamente, a través del paciente intervenido en cirugía ambulatoria, el consejo administrado en relación al tabaquismo y la cirugía. Material y métodos: Estudio monocéntrico, transversal, realizado en un hospital universitario de tercer nivel. Se realizó una encuesta telefónica a pacientes fumadores vistos en la consulta de anestesia. Se evaluaron aspectos relacionados con la actitud del anestesiólogo ante el paciente fumador mediante cuestiones recomendadas para disminuir los riesgos postoperatorios: si advertía de los riesgos del tabaquismo, si aconsejaba dejar de fumar antes de la cirugía y si enviaba al paciente a una unidad de tabaquismo para favorecer la deshabituación. Al finalizar la recogida de datos, se encuestó a los anestesiólogos para evaluar la autopercepción que los mismos reportaban en relación al consejo sanitario a pacientes fumadores. Resultados: Se analizaron 615 consultas hallándose un total de 123 pacientes fumadores (mediana de paquetes año de 12 [rango intercuartil 3,6-23,12]). En relación con la intervención de los anestesiólogos, el 37 % avisó de los riesgos del tabaco y cirugía, un 23 % aconsejó dejar de fumar, y tan solo el 3 % de los mismos envió a los pacientes a una unidad de tabaquismo. En contraste, el 75 % de los anestesiólogos encuestados consideraba que frecuentemente o siempre aconsejaba dejar de fumar antes de la cirugía. Conclusiones: Este estudio muestra que existe una gran diferencia entre las evidencias que muestran la relevancia del abandono del tabaquismo antes de la cirugía y la falta de implementación de las mismas en la práctica habitual durante la consulta de anestesia (AU)

Background: Smokers undergoing surgery, including those experiencing ambulatory surgery are at a higher risk of complications than non-smokers. Preoperative evaluation by an anesthesiologist could provide an excellent opportunity to promote smoking cessation. Previous surveys of anesthesiologists have found that self-reported tobacco counselling rates have room for improvement, however there is no information from the patient’s perspective regarding the smoking cessation interventions in the preoperative ambulatory clinic. Methods: A single-center study was conducted in a tertiary teaching hospital. A telephone survey was realized to all adult cigarette smokers who visited the preoperative ambulatory clinic. The survey recorded the anesthesiologist-delivered interventions to quit smoking before surgery. At the end of the study period, a questionnaire was performed to evaluate the self-reported tobacco counseling of the anesthesiologist Results: Six hundred and fifteen patients were evaluated, of these 123 were current smokers (median packs-years [interquartile range] 12 [3,6-23,12] With regard to preoperative interventions, only 37 % advised patients about the health risks of smoking and 23 % advised them to quit before surgery. Provision of assistance to help the patient to quit was provided in 3 % of cases. In contrast, the self-reported tobacco counseling revealed that 75 % of anesthesiologists stated having advised the patients about the health risks of smoking. Conclusions: This study shows a significant gap between evidence for the advantages of preoperative smoking interventions and implementation in clinical practice. Future studies are badly needed to evaluate the provision of educational materials and other interventions to improve tobacco-counseling rates among anesthesiologists (AU)

Broadly cross-neutralizing antibodies in HIV-1 patients with undetectable viremia.

Several recent studies have identified HIV-infected patients able to produce a broad neutralizing response, and the detailed analyses of their sera have provided valuable information to improve future vaccine design. All these studies have excluded patients on antiretroviral treatment and with undetectable viral loads, who have an improved B cell profile compared to untreated patients. To better understand the induction of neutralizing antibodies in patients on antiretroviral treatment with undetectable viremia, we have screened 508 serum samples from 364 patients (173 treated and 191 untreated) for a broadly neutralizing antibody (bNAb) response using a new strategy based on the use of recombinant viruses. Sera able to neutralize a minipanel of 6 recombinant viruses, including envelopes from 5 different subtypes, were found in both groups. After IgG purification, we were able to confirm the presence of IgG-associated broadly neutralizing activity in 3.7% (7 of 191) of untreated patients with detectable viremia and 1.7% (3 of 174) of aviremic patients receiving antiretroviral treatment. We thus confirm the possibility of induction of a broad IgG-associated neutralizing response in patients on antiretroviral treatment, despite having undetectable viremia. This observation is in stark contrast to the data obtained from long-term nonprogressors, whose little neutralizing activity has been attributed to the low levels of viral replication.

Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon.

The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1-72aa) is sufficient for viral transcript elongation and second exon (73-101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-kappaB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.

c/EBPbeta is a major regulatory element driving transcriptional activation of the CXCL12 promoter.

CXCL12 is considered a constitutively expressed chemokine with homeostatic functions. However, induction of CXCL12 expression and its potential role in several pathologic conditions have been reported, suggesting that CXCL12 gene expression can be induced by different stimuli. To elucidate the molecular mechanisms involved in the regulation of CXCL12 gene expression, we aim to define the molecular factors that operate at the transcriptional level. Basal, constitutive expression of CXCL12 was dependent on basic helix-loop-helix factors. Transcriptional up-regulation of the CXCL12 gene was induced by cellular confluence or inflammatory stimuli such as interleukin-1 and interleukin-6, in a CCAAT/enhancer binding protein beta (c/EBPbeta)-dependent manner. Chromatin immunoprecipitation assays confirmed c/EBPbeta binding to a specific response element located at -1171 of the promoter region of CXCL12. Our data show that c/EBPbeta is a major regulatory element driving transcription of the CXCL12 gene in response to cytokines and cell confluence.

Ellagitannins from Tuberaria lignosa as entry inhibitors of HIV.

Screening of plants from the Iberian Peninsula for anti-human immunodeficiency virus (-HIV) activity revealed that aqueous extract of Tuberaria lignosa gave positive results. Following an activity-guided procedure, the crude extract was counterextracted, and the subsequent fractions obtained tested for their anti-HIV activity in vitro. The bioassay-guided fractionation of the extract afforded an ellagitannin enriched fraction (EEF) isolated for the first time from this species. This EEF exhibited antiviral activity against HIV in MT-2 infected cells, with an IC(50) value of 2.33mug/ml (selectivity index greater than 21). Inhibition of HIV infection by EEF appears to be mediated by CD4 down-regulation, the main receptor for HIV entry. CXCR4 and CCR5 receptors were not affected by EEF, explaining why EEF is able to inhibit R5 and X4 infections.

In vitro analysis of synergism and antagonism of different nucleoside/nucleotide analogue combinations on the inhibition of human immunodeficiency virus type 1 replication.

In this study we have developed an in vitro system to evaluate the combined effect of two NRTIs on HIV replication and to assess their antagonism or synergy. Synergy or antagonism effect was determined in peripheral blood mononuclear cells (PBMCs) to approach a more physiological model than T-cell lines. PBMCs were infected with a full-length HIV-1 clone carrying the luciferase gene as a reporter. The following combinations were investigated: zidovudine+stavudine (ZDV + d4T), lamivudine + abacavir (3TC + ABC), lamivudine + didanosine (3TC + ddI), lamivudine + stavudine (3TC + d4T), tenofovir + stavudine (TDF + d4T), tenofovir + didanosine (TDF + ddI), tenofovir + abacavir (TDF + ABC), tenofovir + lamivudine (TDF + 3TC), tenofovir + zidovudine (TDF + ZDV), stavudine + didanosine (d4T + ddI), zidovudine + lamivudine (ZDV + 3TC), abacavir + didanosine (ABC + ddI), zidovudine + didanosine (ZDV + ddI), and abacavir + stavudine (ABC + d4T). The effect of combining two drugs was evaluated with a quantitative method based on the median-effect principle of Chou and Talalay. A synergistic effect was observed with combinations containing TDF and ZDV or d4T, d4T and ddI and ZDV plus 3TC. In contrast, combinations including TDF + ddI, 3TC + ddI, ABC + ddI, and ZDV + ddI showed an antagonistic effect on the inhibition of viral replication at all levels of inhibition tested. Lower antagonistic effect was also found in drug combinations that included 3TC + ABC, 3TC + TDF, 3TC + d4T, and TDF + ABC. In conclusion, the method developed allows to measure in vitro the effect of different combinations of two NRTIs on HIV replication. The results suggest that combined therapy including TDF with thymidine analogues may be considered for future therapeutic options in contrast to clearly antagonistic combinations such us TDF plus ddI or 3TC plus ddI, that would explain virological failure in clinical studies when these combinations were used.

A new strategy based on recombinant viruses for assessing the replication capacity of HIV-1.

OBJECTIVE: In heavily pretreated patients, resistance mutations arise in both protease (PR) and reverse transcriptase (RT) sequences; however, the relative impact of PR and RT mutations on viral fitness cannot be evaluated with the majority of systems. To address this issue we have developed a model based on recombinant viruses (RVs) that allows the analysis of the replication capacity (RC) of viral populations in which PR and RT are cloned either in combination or separately. METHODS: RVs were generated for full-length polymerase (pol) gene, PR or RT sequences from nine naïve and 14 heavily pretreated HIV-infected patients in therapeutic failure. The relative RC was assessed by comparing luciferase activity between mutant RV and wild-type (wt) isolates. RESULTS: A strong decrease (>60%) in the RC of the pol RV population was observed in the 14 heavily pretreated patients as compared with the wt RVs. The analysis of PR and RT RVs from these patients showed that the decrease in RC was mainly attributable to PR sequences in three of these 14 patients and to RT sequences in seven of these patients. In the four remaining patients, PR and RT sequences independently reduced the RC of the RVs to similar extents. CONCLUSIONS: Different patterns of mutations in either PR or RT have a strong impact on RC in highly experienced HIV-infected patients.

A new strategy based on recombinant viruses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1.

The emergence of drug-resistant variants during antiretroviral therapy is a serious obstacle to sustained suppression of the human immunodeficiency virus type 1 (HIV-1). For that reason, resistance assays are essential to guide clinicians in the selection of optimal treatment regimens. Genotypic assays are less expensive and results are available faster than phenotypic assays. However, in heavily experienced patients with multiple treatment failures interpretation of complex mutation patterns remains difficult, and in these cases phenotypic assays are recommended. This report describes a novel recombinant virus assay where protease (PR) and reverse transcriptase (RT) sequences derived from the plasma isolated from patients are introduced into the back-bone of an HIV molecular clone that expresses Renilla luciferase protein in the place of nef gene. All drug resistance profiles analyzed correlate with previously reported data and showed high reproducibility. This assay, in addition to a fast (completed in 10 days), precise, reproducible and automated method, presents several advantages as compared to other phenotypic assays. The system described below allows the generation of recombinant viruses with multiples cycles of replication carrying a reporter gene in their genomes. These features increase the sensitivity of the test, an important aspect to be considered in the evaluation of less fit viral isolates. In conclusion, the assay permits the quantitation of the level of resistance of clinical HIV-1 isolates to PR and RT inhibitors.

Generation of Human Monoclonal Antibodies from HIV-Infected Individuals / Generación de anticuerpos monoclonales humanos a partir de individuos infectados por VIH

Recently, the protective role of anti-HIV-1 neutralising antibodies has been «re-discovered». Few broadly protective epitopes on the HIV envelope proteins are known, which were defined by few human monoclonal antibodies (mAbs) derived many years ago from non-progressor HIV-infected individuals. It is of interest to try to generate new neutralising human mAbs both to identify new protective epitopes for vaccine design and as potential passive immunotherapy agents. In addition, it is now known that rather than recognising HIV envelope proteins, some neutralising antibodies might be autoantibodies directed to the receptor (CD4) or co-receptors (CCR5 or CXCR4) on the HIV target cells. In that context, we attempted to generate neutralising human mAbs from few HIV-1-infected individuals who, after structured antiretroviral therapy interruptions, showed good virologic and immunologic responses, including serum HIV neutralising activity. We employed the classical heterohybridoma technology and found that the best screening strategy is a primary selection of IgG-producing hybridomas, followed by secondary screenings with different antigens and assays such as HIV neutralisation activity, direct binding to HIV components, binding to HIV target cells, and to cell lines transfected with human HIV receptors and co-receptors. From a single subject, 61 IgG-producing hybridomas out of 5,760 primary wells were obtained, and preliminary data indicate the presence of six (out of 23 tested) neutralising antibodies of the X4 strain, eight anti-p24 antibodies and two antibodies that bind to the MT-2 cell line, the target of X4 strains. None of the 61 mAbs obtained reacted with cells expressing CD4, CXCR4 or CCR5. Other screening tests such as neutralisation assay with the R5 strain, binding to HIV gp120-CD4 covalent complex, and to whole chemically (aldrithiol-2)-inactivated HIV are in progress. The screening strategy is also a means to “open” the repertoire of IgG antibodies of circulating B cells in HIV-infected individuals permitting to assess the frequency of HIV-related IgG antibodies and the IgV genes encoding them, an issue largely unknown (AU)

Recientemente se ha «re-descubierto» el carácter protectivo de los anticuerpos anti-VIH neutralizantes. Se conocen sólo unos pocos epitopos protectivos en las proteínas de la envoltura del VIH definidos gracias a unos pocos anticuerpos monoclonales humanos (mAbs) generados hace ya tiempo en pacientes infectados asintomáticos. Tiene interés intentar generar otros mAbs humanos neutralizantes que puedan definir nuevos epitopos protectivos para el diseño de vacunas, y ser útiles en inmunoterapia pasiva. Además, actualmente está claro que podrían existir anticuerpos neutralizantes que en vez de ir dirigidos contra la envoltura del VIH, reconocieran al receptor (CD4) o coreceptores (CCR5 o CXCR4) en la membrana de las células diana del VIH. En este contexto, hemos intentado obtener mAbs neutralizantes a partir de aquellos pacientes VIH+ que, tras un protocolo de interrupciones estructuradas de la terapia antiretroviral, mostraron buena respuesta virológica e inmunológica, con aumento de actividad sérica neutralizante. Utilizamos la metodología de heterohybridomas convencional y hallamos que la mejor estrategia de escrutinio es la selección primaria de hibridomas secretores de IgG, y luego el escrutinio con distintos ensayos para actividad neutralizante, unión a proteínas víricas, unión a la membrana de las células diana y a células transfectadas con receptores y co-receptores del VIH. A partir de un paciente, de 5.760 microcultivos primarios, se obtuvieron 61 hibridomas productores de IgG entre los que, según datos preliminares, hay seis mAbs (de 23 probados) neutralizantes contra cepas X4, ocho anti-p24 del VIH, y dos que se unen a la componentes de la membrana de la línea MT-2, diana de las cepas X4. Ningún mAb de los 61 obtenidos se unía a CCR5, CD4 o CXCR4. Hay otros escrutinios en curso como neutralización frente a cepa R5, la unión a complejo covalente gp120-CD4 y a VIH total inactivado químicamente con aldritiol-2. El escrutinio utilizado es también un modo de abrir el repertorio de anticuerpos IgG de los linfocitos B circulantes en individuos VIH+ y averiguar la frecuencia de los anticuerpos de especificidad relacionada con el VIH y los genes IgV que los codifican, un aspecto apenas estudiado (AU)

Screening of selected plant extracts for in vitro inhibitory activity on human immunodeficiency virus.

As part of our screening of anti-AIDS agents from natural sources, extracts of 15 medicinal plants widely used in the folk medicines of North America and Europe were evaluated in vitro. Most of the extracts tested were relatively nontoxic to human lymphocytic MT-2 cells, but only the extracts of Hysopp officinalis and Dittrichia viscosa exhibited anti-HIV activity in an in vitro MTT assay. The 50% hydroalcohol extract of Hysopp officinalis and the aqueous extract of Dittrichia viscosa showed inhibitory effects against HIV-1 induced infections in MT-2 cells at concentrations ranging from 50 to 100 microg/mL and 25 to 400 microg/mL, respectively. Both extracts showed no appreciable cytotoxicity at these concentrations.

Herramientas de laboratorio para individualizar el tratamiento: resistencias y niveles de fármacos / Laboratory tools for personalising treatment: resistance and drug levels

A pesar del éxito del tratamiento antirretroviral (TARV) todavía nos enfrentamos a varios problemas relacionados con el mismo, entre los cuales destacan la toxicidad y el fracaso virológico. En relación a este último, aunque las causas del mismo pueden ser muy diversas, la aparición de resistencias va a estar estrechamente correlacionada. La utilización de los tests de resistencias para individualizar el tratamiento ha demostrado ser un instrumento útil para alcanzar el objetivo de la indetectabilidad. Por otro lado, la monitorización de niveles plasmáticos de fármacos, aunque todavía con datos escasos, nos está demostrando que, en situaciones determinadas, va a estar estrechamente ligada con el éxito del tratamiento. Por último, la correlación de las dos técnicas, resistencias y niveles plasmáticos en una sola variable, como es el cociente inhibitorio, parece que en un futuro cercano se convertirá en predictora del éxito del tratamiento de rescate en los pacientes en fracaso (AU)

Despite the success of antiretroviral treatment, we still face various problems related to it, particularly toxicity and virological failure. Although the reasons for the latter may be very diverse, the appearance of resistance correlates very closely. The use of the resistance test to personalise treatment has shown that it is a useful instrument for reaching the objective of being undetectable. In addition, the monitoring of drug plasma levels, though still with scant data, shows us that in determined situations it is going to be intimately linked to the success of the treatment. Finally, the correlation of the two techniques, resistance test and plasma levels, in one sole variable such as the inhibitory quotient may well become in the near future predictive of the success of rescue treatment of patients in failure (AU)

Anti-HIV activity of medicinal plant extracts.

As part of our screening of anti-AIDS agents from natural sources, ethanolic and aqueous extracts of 15 medicinal plants widely used in the folk medicine of the Iberian Peninsula were evaluated in vitro. Most of the extracts tested were relatively nontoxic to human lymphocytic MT-2 cells, but only the extracts of Tuberaria lignosa and Sanguisorba minor magnolii exhibited anti-HIV activity in an in vitro MTT assay. The aqueous extracts of these plants showed inhibitory effects against HIV-1 induced infections in MT-2 cells at concentrations ranging from 12.5 to 50 microg/ml and 50 microg/ml, respectively. Both extracts showed no appreciable cytotoxicity at these concentrations.

The hepatitis B virus X protein induces HIV-1 replication and transcription in synergy with T-cell activation signals: functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 long terminal repeat promoter.

Co-infection with hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1) is relatively common. However, the impact of this co-infection on the clinical outcome of HIV infection has not been elucidated. We herein demonstrate that the HBV X protein (HBx) superinduces ongoing HIV-1 replication and HIV-1 long terminal repeat (LTR) transcription by synergizing with Tat protein and with T-cell activation signals. Although HBx cooperated with mitogenic stimuli in the induction of reporter plasmids harboring the HIV-1 kappaB enhancer, in both a NF-kappaB-dependent manner and a NF-AT-dependent manner, deletion of this element from the LTR did not affect the HBx-mediated up-regulation in the presence of Tat and/or mitogens. In contrast, mutation of the proximal LTR Sp1-binding sites abolished the HBx-mediated synergistic activation, but only when it was accompanied by deletion of the kappaB enhancer. When HBx was targeted to the nucleus, its ability to synergize with cellular activation stimuli was maintained. Furthermore, mutations of HBx affecting its interaction with the basal transcription machinery abrogated the synergistic activation by HBx, suggesting that this protein exerts its function by acting as a nuclear co-activator. These results indicate that HBx could contribute to a faster progression to AIDS in HBV-HIV co-infected individuals.