Objectives: Antiretroviral (ARV) drugs have played a vital role in controlling the HIV-1 epidemic; however, some challenges remain. ARV drugs vary in their ability to control HIV infection, displaying differences in treatment-limiting factors and genetic barriers to resistance. The current report assesses the prevalence of HIV-1 drug resistance mutations (DRMs) among patients who failed first-line antiretroviral therapy (ART) and evaluates the genetic barrier of different regimens.Methods: The study cohort ( n = 271) included HIV-infected individuals who visited the Novosibirsk, Russia, HIV/AIDS clinic in 2018-2022. All patients received first-line ART prior to virological failure. Sociode-mographic and HIV-related data were collected from medical records and self-reported questionnaires. HIV-1 pol gene sequences were generated, and the presence of HIV-1 DRM was assessed. The genetic barrier to resistance was assessed by combining treatment regimen and adherence data.Results: Nonoptimal ART adherence was identified in 48.3% of patients and correlated with male sex, PWID, unemployment, and rural area residence. Most of the patients with high-level adherence were identified among those who were on TDF + 3TC + DTG. HIV-1 DRMs were identified in 54.6% of the patients. The analysis of HIV-1 DRM, ART regimen, and adherence data classified TDF + 3TC + DTG and TDF + 3TC + LPV/r as treatment regimens with a high genetic barrier, whereas EFV-containing ART was classified as a regimen with a low genetic barrier.Conclusions: The current study delivers results on the efficacy of HIV-1 ART and treatment adherence in real-world practice settings. This report suggests that ART regimens with a high genetic barrier to resistance combined with improved treatment adherence may reduce the transmission of HIV-1 resistant variants.(c) 2023 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

   Aim. To research the molecular epidemiological features of the territorial spread of HIV infection and the influence of recombination variability on the formation of the HIV‐1 population in the Primorsky Krai, Russia.   Materials and Methods. A comprehensive analysis of the molecular genetic characteristics of HIV‐1 and clinical‐epidemiological data of 127 HIV‐infected residents of Primorsky Krai was performed. The nucleotide sequences of the pol gene region encoding protease, reverse transcriptase and integrase of HIV‐1 were obtained by the sequencing of amplified fragments of the virus. Genotyping, phylogenetic, and mutational analyses were performed using specialised software resources.   Results. In the samples studied of HIV‐1 isolated in the Primorsky Krai, the dominance of HIV‐1 subtype A6 was detected as 40.9 %. The prevalence of subtype B viruses is high compared to other Russian regions – 29.9 %. The HIV‐1 subtype C was found in 6.3 % of the samples studied; circulating recombinant forms CRF63_02A6 – 6.3 %, CRF02_AG – 0.8 %. Unique viruses resulting from the recombination of HIV subtypes A6 and B, A6 and C, C and B were found in 15.7 % of cases (URF_A6C, URF_CB, URF_BA6). Of the 18 HIV‐1 URFs described, 14 were variants of URF_A6C (11 %). Analysis of the near full‐length genome (NFLG) of URF_A6C made it possible to find and describe a new HIV genovariant spreading in Primorye ‐ CRF157_A6C.   Conclusion. The analysis of HIV‐1 circulating in the Primorsky Krai confirmed that a specific molecular epidemic situation is developing in the region, which is characterized by active transmission of viruses not only of subtype A6, but also of subtypes B and C, which is not observed in other regions of Russia. For the first time in the Far East, a high frequency of HIV‐1 new unique recombinant forms occurrence has been described, some variants of which are beginning to spread among residents of the Primorsky Krai. An increase in the genetic heterogeneity of the viral population due to recombination variability of HIV is a negative prognostic factor for the territorial epidemic progress of HIV infection in the Far East.

This study investigated the antiretroviral efficacy, toxicity profile, and cellular uptake of LNA-modified oligonucleotides within an in vitro HIV infection model. Phosphorothioate (PS) oligonucleotides, designed to bind conserved regions of the HIV-1 genome, were modified at the 3′ and/or 5′ ends with LNA nucleotides. The antiviral properties of oligonucleotides against HIV-1 subtype A6 were evaluated using human MT-4 cell cultures. The antiretroviral activity of LNA-oligonucleotides against HIV-1 has been established. Variations in the 50% inhibitory viral reproductive dose (IC50) values among the oligonucleotides were observed, depending upon both the target and the incorporated LNA modification. The optimal IC50 values (90 ± 10 nM) were achieved using a PS oligonucleotide lacking LNA modifications, which targeted the HIV-1 integrase-encoding genomic region. Optimal IC50 values (90 ± 10 nM) were achieved using a PS oligonucleotide lacking LNA modifications, which targeted the HIV-1 integrase-encoding genomic region. Optimal HIV inhibitory action among LNA constructs was observed in an oligonucleotide with a 5′-end LNA modification targeting the HIV integrase region (IC50 = 1.12 ± 0.03 μM). The introduction of LNA modifications to PS oligonucleotides failed to enhance antiviral activity, as demonstrated by IC50 values revealing significant in vitro HIV-1 inhibitory capacity. The internalization of oligonucleotides demonstrating optimal IC50 values was investigated via flow cytometry and imaging techniques. The PS modification has been demonstrated to exhibit the highest penetration efficiency. The characteristic features included low toxicity (maintaining >92% viable cells after 48 hours of culture), high cytoplasmic membrane sorption capacity (approximately 12% FAM+ cells after 48 hours), high penetration efficiency (approximately 98% FAM+ cells showing cytoplasmic signal), and elevated internalization and entropy ratios.

This study investigated the antiretroviral efficacy, toxicity profile, and cellular uptake of phosphothioate (PS) and PS/LNA-modified oligonucleotides within an in vitro HIV infection model. Phosphothioate (PS) oligonucleotides, designed to bind conserved regions of the HIV-1 genome, were modified at the 3’ and/or 5’ ends with LNA nucleotides. The antiviral properties of oligonucleotides against HIV-1 subtype A6 were evaluated using human MT-4 cell cultures. The antiretroviral activity of LNA-oligonucleotides against HIV-1 has been established. Variations in the 50% inhibitory viral reproductive dose (IC50) values among the oligonucleotides were observed, depending upon both the target and the incorporated LNA modification. Optimal IC50 values (90 ± 10 nM) were achieved using a PS oligonucleotide lacking LNA modifications, which targeted the HIV-1 integrase-encoding genomic region. Optimal HIV inhibitory action among LNA constructs was observed in an oligonucleotide with a 5’-end LNA modification targeting the HIV integrase region (IC50 = 1.12 ± 0.03 μM). The introduction of LNA modifications to PS oligonucleotides failed to enhance antiviral activity, as demonstrated by IC50 values revealing significant in vitro HIV-1 inhibitory capacity. The internalization of oligonucleotides demonstrating optimal IC50 values was investigated via flow cytometry and imaging techniques. Antisense oligonucleotides with single PS modification showed better antiretroviral activity with lower IC50 value compared to PS/LNA-modified antisense oligonucleotides. The reason for this difference is the better internalization ability of PS-modified oligonucleotides. The characteristic features included low toxicity (maintaining >92% viable cells after 48 h of culture), high cytoplasmic membrane sorption capacity (approximately 12% FAM + cells after 48 h), high penetration efficiency (approximately 98% FAM + cells showing cytoplasmic signal), and elevated internalization and entropy ratios.

For many therapeutic agents to be effective against intracellular targets, they must first be able to penetrate the cell membrane. Current methodologies for assessing internalization, such as confocal microscopy and conventional flow cytometry, are limited by low throughput or an inability to provide precise spatial information on signal localization. Here, we present a comprehensive, semi-automated analytical pipeline for investigating compound internalization based on imaging flow cytometry, which is designed to address these limitations. Our workflow details the procedure from sample preparation and data acquisition on an Amnis FlowSight cytometer to analysis using IDEAS 6.2 software with a custom-designed template. Key features of our approach include the automated discrimination of signal between the plasma membrane and cytoplasmic compartments, the calculation of an internalization coefficient, and the introduction of a novel parameter—signal distribution entropy—to quantify the uniformity of the compound distribution within cells. For the statistical analysis, we developed FluoSta v1.0, a software tool that automates descriptive statistics and analysis of variance (ANOVA with Tukey’s post hoc test) and facilitates data visualization. The pipeline’s utility was demonstrated in a series of model experiments, including a comparative assessment of the internalization efficiency of PS- versus PS/LNA-modified compounds in MT-4 cell cultures.

Aim. To investigate the cell entry and antiretroviral properties of modified oligodeoxyribonucleotides targeting highly conserved regions of the HIV‐1 genome: the study was conducted using modified oligonucleotides containing phosphorothioate, phosphorylguanidine or dodecyl moieties.The ability of oligonucleotides to inhibit the virus was studied using a model of lymphoid culture of human MT4 cells infected with a characterised high‐productive strain of HIV‐1 belonging to the subtype A6 genovariant widely spread in the Russian Federation. The entry of oligonucleotides into MT4 cells was assessed by confocal microscopy.It was shown that phosphorothioate‐ and dodecyl‐containing oligonucleotides are able to penetrate inside cells without the use of any additional transfection agents. It was found that phosphorothioate oligonucleotides transfected in duplex with dodecyl‐containing derivatives were localized inside the cell in the nucleus and nucleolus and were able to inhibit the reproduction of a highly productive HIV‐1 strain. A 50% toxic dose (TC50) and a 50% inhibitory concentration (IC50) were determined for the oligonucleotide derivatives studied, the value of which was less than 0,5 μM in the case of phosphorothioate oligonucleotides.The data obtained indicate the ability of the modified oligonucleotides studied to be used as potential antiviral agents against HIV‐1.