Effectiveness and safety of integrase strand transfer inhibitors in Spain: a prospective real-world study

HIV; Integrase strand transfer inhibitorsVIH; Inhibidores de transferencia de cadena de integrasaVIH; Inhibidors de transferència de cadena de la integrasaIntroduction: Second-generation integrase strand transfer inhibitors (INSTIs) are preferred treatment options worldwide, and dolutegravir (DTG) is the treatment of choice in resource-limited settings. Nevertheless, in some resource-limited settings, these drugs are not always available. An analysis of the experience with the use of INSTIs in unselected adults living with HIV may be of help to make therapeutic decisions when second-generation INSTIs are not available. This study aimed to evaluate the real-life effectiveness and safety of dolutegravir (DTG), elvitegravir/cobicistat (EVG/c), and raltegravir (RAL) in a large Spanish cohort of HIV-1-infected patients. Methods: Real-world study of adults living with HIV who initiated integrase INSTIs DTG, EVG/c, and RAL-based regimens in three settings (ART-naïve patients, ART-switching, and ART-salvage patients). The primary endpoint was the median time to treatment discontinuation after INSTI-based regimen initiation. Proportion of patients experiencing virological failure (VF) (defined as two consecutive viral loads (VL) ≥200 copies/mL at 24 weeks or as a single determination of VL ≥1,000 copies/mL while receiving DTG, EVG/c or RAL, and at least 3 months after INSTI initiation) and time to VF were also evaluated. Results: Virological effectiveness of EVG/c- and RAL-based regimens was similar to that of DTG when given as first-line and salvage therapy. Treatment switching for reasons other than virological failure was more frequent in subjects receiving EVG/c and, in particular, RAL. Naïve patients with CD4+ nadir <100 cells/μL were more likely to develop VF, particularly if they initiated RAL or EVG/c. In the ART switching population, initiation of RAL and EVG/c was associated with both VF and INSTI discontinuation. There were no differences in the time to VF and INSTI discontinuation between DTG, EVG/c and RAL. Immunological parameters improved in the three groups and for the three drugs assessed. Safety and tolerability were consistent with expected safety profiles. Discussion: Whereas second-generation INSTIs are preferred treatment options worldwide, and DTG is one of the treatment of choices in resource-limited settings, first-generation INSTIs may still provide high virological and immunological effectiveness when DTG is not available.This study received funding from ViiV Healthcare and Fight Infections Foundation. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication

Atomic Force Microscopy and Voltammetric Investigation of Quadruplex Formation between a Triazole-Acridine Conjugate and Guanine-Containing Repeat DNA Sequences

The interactions of the Tetrahymena telomeric repeat sequence d(TG4T) and the polyguanylic acid (poly(G)) sequence with the quadruplex-targeting triazole-linked acridine ligand GL15 were investigated using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite and voltammetry at a glassy carbon electrode. GL15 interacted with both sequences, in a time dependent manner, and G-quadruplex formation was detected. AFM showed the adsorption of quadruplexes as small d(TG4T) and poly(G) spherical aggregates and large quadruplex-based poly(G) assemblies, and voltammetry showed the decrease and disappearance of GL15 and guanine oxidation peak currents and appearance of the G-quadruplex oxidation peak. The GL15 molecule strongly stabilized and accelerated G-quadruplex formation in both Na+ and K+ ion-containing solution, although only K+ promoted the formation of perfectly aligned tetra-molecular G-quadruplexes. The small-molecule complex with the d(TG4T) quadruplex is discrete and approximately globular, whereas the G-quadruplex complex with poly(G) is formed at a number of points along the length of the polynucleotide, analogous to beads on a string.Financial support from Fundação para a Ciência e Tecnologia (FCT), Grant SFRH/BPD/92726/2013 (A.-M. Chiorcea-Paquim), Project Grant (A.D.R. Pontinha), projects PTDC/SAU-BMA/118531/2010, PTDC/QEQ-MED/0586/2012, PEst-C/EME /UI0285/2013 and CENTRO-07-0224-FEDER-002001 (MT4MOBI) (co-financed by the European Community Fund FEDER), FEDER funds through the program COMPETE - Programa Operacional Factores de Competitividade is gratefully acknowledged. Work in the S.N. laboratory was supported by Programme Grant No. C129/A4489, from Cancer Research UK, and by the FP6 framework grant “Molecular Cancer Medicine” from the EU. S.S. was a Maplethorpe Fellow of The University of London.Peer reviewe

Whole-Genome Sequencing of Two Bartonella bacilliformis Strains

Bartonella bacilliformis is the causative agent of Carrion's disease, a highly endemic human bartonellosis in Peru. We performed a whole-genome assembly of two B. bacilliformis strains isolated from the blood of infected patients in the acute phase of Carrion's disease from the Cusco and Piura regions in Peru

Decreased Phenotypic Susceptibility to Etravirine in Patients with Predicted Genotypic Sensitivity

A sensitive, phenotypic reverse transcriptase (RT)-based drug susceptibility assay for the detection of etravirine (ETR) resistance in patient isolates was developed and compared with the results from direct sequencing and ultra-deep pyrosequencing (UDPS). Samples were obtained from 15 patients with antiretroviral therapy (ART) failure and from five non-nucleoside reverse transcriptase inhibitor (NNRTI)-naïve patients of whom four were infected by an NNRTI-resistant strain (transmitted drug resistance, TDR). In five patients, two consecutive samples (a and b) were taken for follow up of the virological response. HIV-1 RT was purified and drug susceptibility (IC) to ETR was estimated. Direct sequencing was performed in all samples and UDPS in samples from nine patients. Increased IC to ETR was found in samples from 13 patients where direct sequencing predicted resistance in only four. UDPS identified additional (N = 11) NNRTI resistance associated mutations (RAMs) in six of nine tested patients. During early failure, IC increases were observed in three of six patients without any ETR-RAMs detected by direct sequencing. In further two patients, who stopped NNRTI before sampling, increased IC values were found shortly after, despite absence of ETR-RAMs. In two patients who had stopped NNRTI for >1 year, a concordance between phenotype and genotypes was found. Two patients with TDR had increased IC despite no ETR-RAMs were detected by direct sequencing. UDPS revealed additional ETR-RAMs in four patients with a discrepancy between phenotype and direct sequencing. The RT-based phenotypic assay showed decreased ETR susceptibility in patients where direct sequencing predicted ETR-sensitive virus. This increased phenotypic sensitivity was to a large extent supported by UDPS and treatment history. Our method could be valuable for further studies on the phenotypic kinetics of NNRTI resistance. The clinical relevance remains to be studied in larger patient-populations

Vaccination with an HIV T-cell immunogen induces alterations in the mouse gut microbiota

The gut microbiota is emerging as a crucial factor modulating vaccine responses; however, few studies have investigated if vaccines, in turn, can alter the microbiota and to what extent such changes may improve vaccine efficacy. To understand the effect of T-cell vaccination on the gut microbiome, we administered an HIV-1 T-cell immunogen (HTI arm) or PBS (control, Mock arm) to C57Bl/6 mice following a heterologous prime-boost scheme. The longitudinal dynamics of the mice gut microbiota was characterized by 16 S ribosomal RNA sequencing in fecal samples collected from cages, as well as from three gut sections (cecum, small and large intestine). Serum and spleen cells were obtained at the last time point of the study to assess immune correlates using IFNγ ELISPOT and cytokine Luminex ® assays. Compared with Mock, HTI-vaccinated mice were enriched in Clostridiales genera (Eubacterium xylanophilum group, Roseburia and Ruminococcus) known as primary contributors of anti-inflammatory metabolites, such as short-chain fatty acids. Such shift was observed after the first HTI dose and remained throughout the study follow-up (18 weeks). However, the enriched Clostridiales genera were different between feces and gut sections. The abundance of bacteria enriched in vaccinated animals positively correlated with HTI-specific T-cell responses and a set of pro-inflammatory cytokines, such as IL-6. This longitudinal analysis indicates that, in mice, T-cell vaccination may promote an increase in gut bacteria known to produce anti-inflammatory molecules, which in turn correlate with proinflammatory cytokines, suggesting an adaptation of the gut microbial milieu to T-cell-induced systemic inflammation

Next-Generation Virus Genotyping for HIV-1 Surveillance and Clinical Management

Tot i que la infecció pel virus de la immunodeficiència humana de tipus 1 (VIH-1) roman sense cura, la teràpia antiretroviral és capaç de bloquejar de manera persistent la replicació viral, limitant el dany al sistema immunitari. Aquest fet pot prevenir i/o revertir el deteriorament immunitari en la majoria de pacients, prolongant la seva esperança de vida i incrementant la seva qualitat. A més, el tractament antiretroviral és la eina disponible més eficaç per prevenir la posterior transmissió del virus. Una de les qüestions més crítiques en el tractament del VIH-1 son les mutacions de resistència. El VIH-1 es distribueix en quasispecies, fet que permet al virus adaptar-se i escapar ràpidament de l'efecte dels fàrmacs o de la pressió immunitària. També implica que mutacions minoritàries que poden ser potencialment rellevants també son presents, i poden passar desapercebudes pels assajos genotípics convencionals. En altres paraules, els metges poden perdre informació important necessària per optimitzar les opcions de tractament antiretroviral. Mitjançant tècniques de seqüenciació massiva hem demostrat que les mutacions minoritàries de resistència als antiretrovirals poden ser clínicament importants en alguns pacients i quan es prescriuen règims antiretrovirals concrets. Vam trobar que les mutacions de resistència minoritàries pre-existents son clínicament rellevants en pacients que es presenten tard a la clínica amb una supressió immunològica avançada que comencen tractament amb inhibidors de la transcriptasa reversa no anàlegs de nucleòsids. La seva presència s'associa amb més risc de fracàs virològic. També hem observat que la presència d'aquestes mutacions minoritàries pot ser important pels individus infectats per VIH-1 que viuen en regions en vies de desenvolupament, on els pacients sovint son tractats amb règims de primera línia que contenen tenofovir sense un posterior monitoreig de la càrrega viral. Vam trobar que la prevalença de mutacions de resistència a tenofovir després d'un fracàs virològic és alta, i l'absència de genotipat podria comprometre la utilització del tenofovir com a règim de segona línia. Per tant, la vigilància de les resistències a tenofovir hauria de ser una prioritat tant en pacients naïve a tractament com en pacients experimentats, com a mínim mitjançant seqüenciació poblacional. Un altre missatge important és que fins a dia d'avui la transmissió de mutacions de resistència als inhibidors de la integrasa a Europa roman a nivells mínims. Ara que s'han convertit en una família de tractament que es prescriu freqüentment en països desenvolupats, podria portar a l'emergència de mutacions de resistència transmeses i a una necessitat de genotipar per les mutacions a integrasa en els propers anys. Pel que fa a l'anàlisi de la presència de la mutació de resistència S282T en pacients co-infectats per VIH-1/hepatitis C, no la vam detectar en cap cas, i globalment s'ha vist en molt pocs pacients, amb una reversió a forma wild-type passades unes setmanes. A més, amb els nous règims anomenats agents antivirals directes els pacients es curen en la majoria de casos, sense necessitat de testar les mutacions de resistència abans d'administrar-los. En general, la detecció de resistències minoritàries als fàrmacs pot ajudar a evitar fracassos virològics i prevenir la transmissió de variants resistents de VIH.Although HIV-1 infection cannot be cured, antiretroviral therapy is able to persistently block virus replication, limiting the damage on the immune system. This can prevent and/or revert immune deterioration in most subjects, prolonging life expectancy and increasing its quality. Furthermore, antiretroviral treatment is the most effective tool available to prevent onward HIV-1 transmission. One of the most critical issues in HIV-1 treatment is resistance mutations. HIV-1 is distributed in quasispecies, which allows the virus to rapidly adapt and escape from adverse drug or immune pressure. It also implies that potentially relevant low-frequency mutants exist, which may go unnoticed by conventional genotypic assays. In other words, clinicians might miss important information to optimize antiretroviral treatment choices. By using ultrasensitive sequencing techniques we have demonstrated that low frequency drug resistance mutations may be clinically important in certain patients and when prescribing particular antiretroviral regimens. We found that pre-existing low frequency drug resistance mutations are clinically relevant in subjects who present late to clinics with advanced immune suppression, who start treatment with non-analogue reverse transcriptase inhibitors, as its presence is associated with higher risk of virological failure. We have also observed that the presence of these minority mutations may be important for HIV-1 infected people living in low and middle income countries, where patients are often given first-line tenofovir containing regimens with no virological monitoring. We found that the prevalence of tenofovir resistance after virological failure is high, and absence of genotyping could compromise tenofovir use as second-line regimen. Therefore, surveillance of tenofovir resistance should be a priority in treated and naïve populations, at least by population sequencing. Another important message is that, to date, transmission of integrase strand transfer inhibitors (INSTIs) resistance in Europe remains at negligible levels. Now that they are frequently being prescribed in resource rich settings, this could lead to the emergence of transmitted resistance mutations and the need for integrase genotyping in the coming years. Regarding the analysis of the presence of the resistance mutation S282T in HIV-1/HCV co-infected patients, we did not detect it, and globally it has only been observed in few patients, with reversion to wild-type viruses within several weeks. Also, prescribing new direct antiviral agents result in HCV cure in the majority of patients, with no need to test for drug resistance mutations before administration. In general, detection of minority drug resistance may help to avoid virological failures and to prevent the transmission of drug resistant HIV

Probiotic effects on immunity and microbiome in HIV-1 discordant patients

Some HIV-1 infected patients are unable to completely recover normal CD4+ T-cell (CD4+) counts after achieving HIV-1 suppression with combined Antiretroviral Therapy (cART), hence being classified as immuno-discordant. The human microbiome plays a crucial role in maintaining immune homeostasis and is a potential target towards immune reconstitution. RECOVER (NCT03542786) was a double-blind placebo-controlled clinical trial designed to evaluate if the novel probiotic i3.1 (AB-Biotics, Sant Cugat del Vallès, Spain) was able to improve immune reconstitution in HIV-1 infected immuno-discordant patients with stable cART and CD4+ counts <500 cells/mm3. The mixture consisted of two strains of L. plantarum and one of P. acidilactici, given with or without a fiber-based prebiotic. 71 patients were randomized 1:2:2 to Placebo, Probiotic or probiotic + prebiotic (Synbiotic), and were followed over 6 months + 3-month washout period, in which changes on systemic immune status and gut microbiome were evaluated. Primary endpoints were safety and tolerability of the investigational product. Secondary endpoints were changes on CD4+ and CD8+ T-cell (CD8+) counts, inflammation markers and faecal microbiome structure, defined by alpha diversity (Gene Richness), beta diversity (Bray-Curtis) and functional profile. Comparisons across/within groups were performed using standard/paired Wilcoxon test, respectively. Adverse event (AE) incidence was similar among groups (53%, 33%, and 55% in the Placebo, Probiotic and Synbiotic groups, respectively, the most common being grade 1 digestive AEs: flatulence, bloating and diarrhoea. Two grade 3 AEs were reported, all in the Synbiotic group: abdominal distension (possibly related) and malignant lung neoplasm (unrelated), and 1 grade 4 AE in the Placebo: hepatocarcinoma (unrelated). Synbiotic exposure was associated with a higher increase in CD4+/CD8+ T-cell (CD4/CD8) ratio at 6 months vs baseline (median=0.76(IQR=0.51) vs 0.72(0. 45), median change= 0.04(IQR=0.19), p = 0.03). At month 9, the Synbiotic group had a significant increase in CD4/CD8 ratio (0.827(0.55) vs 0.825(0.53), median change = 0.04(IQR=0.15), p= 0.02) relative to baseline, and higher CD4+ counts (447 (157) vs. 342(73) counts/ml, p = 0.03), and lower sCD14 values (2.16(0.67) vs 3.18(0.8), p = 0.008) than Placebo. No effect in immune parameters was observed in the Probiotic arm. None of the two interventions modified microbial gene richness (alpha diversity). However, intervention as categorical variable was associated with slight but significant effect on Bray-Curtis distance variance (Adonis R2 = 0.02, p = 0.005). Additionally, at month 6, Synbiotic intervention was associated with lower pathway abundances vs Placebo of Assimilatory Sulphate Reduction (8.79·10 -6 (1.25·10 -5) vs. 1.61·10 -5 (2.77·10 -5), p = 0.03) and biosynthesis of methionine (2.3·10 -5 (3.17·10 -5) vs. 4·10 -5 (5.66·10 -5), p = 0.03) and cysteine (1.83·10 -5 (2.56·10 -5) vs. 3.3·10 -5 (4.62·10 -5), p = 0.03). At month 6, probiotic detection in faeces was associated with significant decreases in C Reactive Protein (CRP) vs baseline (11.1(22) vs. 19.2(66), median change= -2.7 (13.2) ug/ml, p = 0.04) and lower IL-6 values (0.58(1.13) vs. 1.17(1.59) ug/ml, p = 0.02) when compared with samples with no detectable probiotic. No detection of the probiotic was associated with higher CD4/CD8 ratio at month 6 vs baseline (0.718(0.57) vs. 0.58(0.4), median change = 0.4(0.2), p = 0.02). After washout, probiotic non-detection was also associated with a significant increase in CD4+ counts (457(153) vs. 416(142), median change = 45(75), counts/ml, p = 0.005) and CD4/CD8 ratio (0.67(0.5) vs 0.59(0.49), median change = 0.04 (0.18), p = 0.02). A synbiotic intervention with L. plantarum and P. acidilactici was safe and led to small increases in CD4/CD8 ratio and minor reductions in sCD14 of uncertain clinical significance. A probiotic with the same composition was also safe but did not achieve any impact on immune parameters or faecal microbiome composition

Evolution of the gut microbiome following acute HIV-1 infection

Background: In rhesus macaques, simian immunodeficiency virus infection is followed by expansion of enteric viruses but has a limited impact on the gut bacteriome. To understand the longitudinal effects of HIV-1 infection on the human gut microbiota, we prospectively followed 49 Mozambican subjects diagnosed with recent HIV-1 infection (RHI) and 54 HIV-1-negative controls for 9–18 months and compared them with 98 chronically HIV-1- infected subjects treated with antiretrovirals (n = 27) or not (n = 71). Results: We show that RHI is followed by increased fecal adenovirus shedding, which persists during chronic HIV-1 infection and does not resolve with ART. Recent HIV-1 infection is also followed by transient non-HIV-specific changes in the gut bacterial richness and composition. Despite early resilience to change, an HIV-1-specific signature in the gut bacteriome—featuring depletion of Akkermansia, Anaerovibrio, Bifidobacterium, and Clostridium—previously associated with chronic inflammation, CD8+ T cell anergy, and metabolic disorders, can be eventually identified in chronically HIV-1-infected subjects. Conclusions: Recent HIV-1 infection is associated with increased fecal shedding of eukaryotic viruses, transient loss of bacterial taxonomic richness, and long-term reductions in microbial gene richness. An HIV-1-associated microbiome signature only becomes evident in chronically HIV-1-infected subjects

The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a highmobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4- dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria

Gut microbiome signatures linked to HIV-1 reservoir size and viremia control

The potential role of the gut microbiome as a predictor of immune-mediated HIV-1 control in the absence of antiretroviral therapy (ART) is still unknown. In the BCN02 clinical trial, which combined the MVA.HIVconsv immunogen with the latency-reversing agent romidepsin in early-ART treated HIV-1 infected individuals, 23% (3/13) of participants showed sustained low-levels of plasma viremia during 32 weeks of a monitored ART pause (MAP). Here, we present a multi-omics analysis to identify compositional and functional gut microbiome patterns associated with HIV-1 control in the BCN02 trial. Viremic controllers during the MAP (controllers) exhibited higher Bacteroidales/Clostridiales ratio and lower microbial gene richness before vaccination and throughout the study intervention when compared to non-controllers. Longitudinal assessment indicated that the gut microbiome of controllers was enriched in pro-inflammatory bacteria and depleted in butyrate-producing bacteria and methanogenic archaea. Functional profiling also showed that metabolic pathways related to fatty acid and lipid biosynthesis were significantly increased in controllers. Fecal metaproteome analyses confirmed that baseline functional differences were mainly driven by Clostridial es. Participants with high baseline Bacteroidales/Clostridiales ratio had increased pre-existing immune activation-related transcripts. The Bacteroidales/Clostridiales ratio as well as host immune-activation signatures inversely correlated with HIV-1 reservoir size. The present proof-of-concept study suggests the Bacteroidales/Clostridiales ratio as a novel gut microbiome signature associated with HIV-1 reservoir size and immune-mediated viral control after ART interruption. The online version contains supplementary material available at 10.1186/s40168-022-01247-6