Ludmila Gennadievna Gotfrid

HIV-1 tropism toward the CCR5 and CXCR4 coreceptors is an important viral characteristic associated with cell entry, the potential use of specific entry inhibitors, studies of disease progression, and analyses of proviral reservoirs. The development of tools for genotypic tropism prediction also requires annotated sequence datasets with reliable phenotypic annotation. In this study, we describe a curated HIV-1 sequence dataset intended to complement, rather than replace, existing reference collections. Publications and GenBank records were searched manually using PubMed, Google Scholar, OpenAlex, cross-references, and records from the GenBank Nucleotide database. Sequences were included if a GenBank accession number could be linked to the V3 region of the HIV-1 env gene, or to a longer fragment containing this region, and to phenotypically determined tropism; records already present in LANL were removed. The final dataset comprises 1809 records associated with 32 unique PubMed-indexed publications. Subtype B predominated in the dataset; however, non-B variants accounted for 455 records (25.2%), including several Circulating Recombinant Forms (CRFs) and Unique Recombinant Forms (URFs). R5 variants accounted for 1528 records (84.5%), whereas X4 and R5/X4 variants accounted for 281 records (15.5%). The dataset includes 14 categories of phenotypic assays, geographic metadata, and sequences ranging from env gene fragments to near-complete HIV-1 genomes. This resource can be used as a complement to existing databases for the development, testing, and external validation of approaches for HIV-1 tropism prediction.

Determining HIV-1 tropism provides the prognosis of HIV infection and is required before prescribing maraviroc, an entry inhibitor that blocks the interaction between the viral gp120 and the CCR5 coreceptor. However, existing prediction algorithms have been developed primarily for the globally most prevalent subtypes (B, C, and CRF01_AE) and often show reduced performance for other HIV-1 genetic variants. Sub-subtype A6 and circulating recombinant form CRF63_02A6 dominate the HIV-1 epidemic in Russia and other Former Soviet Union (FSU) countries, yet the reliability of tropism prediction for these viruses remains virtually unexplored. We phenotypically determined the tropism of 25 clinical isolates (11 R5, 1 X4, and 7 dual-tropic R5/X4) using U87.CD4.CCR5 and U87.CD4.CXCR4 cell lines and performed a comparative analysis of eight existing genotypic tools (Geno2pheno, WebPSSM, T-CUP 2.0, the Delobel/Garrido rules, and others) or their modifications on a combined dataset that included Los Alamos National Laboratory (LANL) reference sequences (subtypes A, B, C, CRF01_AE, and CRF02_AG) and our laboratory-derived isolates. Most models achieved high accuracy for globally prevalent subtypes (≈95% for B, C, and CRF01_AE) but showed markedly reduced performance for sub-subtype A6 (best accuracy among existing models, 85%) and CRF63_02A6 (best accuracy, 72%), with a poor balance between sensitivity and specificity. To address this problem, we developed HIV-V3Augur, an ensemble stacking model based on the Random Forest and Support Vector Machine (SVM) machine learning algorithms, trained on Pseudo Amino Acid Composition (PseAAC) and Relative Synonymous Codon Usage (RSCU) features with 10-fold stratified cross-validation. HIV-V3Augur achieved an accuracy of 77%, sensitivity of 79%, and specificity of 79% on sub-subtype A6, and on CRF63_02A6 it reached an accuracy of 95%, sensitivity of 87%, and specificity of 100%. Cross-validation demonstrated that HIV-V3Augur represents a balanced genotypic tropism prediction tool for understudied HIV-1 variants circulating in the FSU region. HIV-V3Augur can be used locally through a graphical user interface.

The goal of this study was to investigate the effectiveness in suppressing viral replication of combinations of antisense phosphorothioate oligodeoxyribonucleotides targeting conserved regions in the HIV‐1 genome. The antiretroviral activity of the antisense oligonucleotides was evaluated using human MT4 lymphocyte culture infected with HIV‐1 subtype A6. The amount of viral protein p24 was measured using an enzyme immunoassay (ELISA) to determine the effectiveness of the oligonucleotides treatments.The oligonucleotides studied and their combinations showed the ability to inhibit HIV‐1 replication at nanomolar concentrations. Among the mono‐oligonucleotides, the one targeting the gag gene region had the best anti‐viral suppression (IC50 = 58 ± 4.4 nM). Among the combinations, the most effective (IC50 = 29 ± 1.7 nM) consisted of oligonucleotides targeting the gag region and the primer binding site. These results confirm the potential of combining oligonucleotides that target different regions of the HIV‐1 genome to synergistically increase the anti‐viral effect, as compared to using each oligonucleotide individually. The justification for using a combination of oligonucleotides is important because the multidirectional antiviral effect of these molecules minimises the potential negative impact of HIV's mutational variability on the effectiveness of antiviral drugs.

We present the single-cell transcriptomic analysis of peripheral blood mononuclear cells (PBMC) from individuals during acute HIV-1 infection caused by viral strains circulating in Russia and the Former Soviet Union (FSU) countries. Using 10x Genomics single-cell RNA sequencing (scRNA-seq) on the Illumina NextSeq 550 platform, we have analyzed scRNA-seq data from three treatment-naive patients (viral load > 1 × 10⁶ copies/mL, estimated infection duration ≤ 4 weeks) and three healthy donors. Data integration (Seurat, Harmony), automated cell-type annotation (CellTypist), and GeneOntology (GO) enrichment analysis for highly expressed and low-expressed genes revealed a profound reorganization of transcriptional programs across key immune populations, including memory CD4⁺ and CD8⁺ T cells, non-classical monocytes and natural killer cells (NK-cells). We observed signatures of hyperactivation of pro-inflammatory pathways (NF-kB, TNF, and type I/II interferon signaling), upregulation of genes associated with cellular migration (CXCR4, CCR7) and metabolic adaptation (oxidative phosphorylation components), alongside a mixed pro- and anti-apoptotic expression profile. Notably, our data pointed to a pronounced dysregulation of the TGF-β and mTOR signaling cascades, disrupted intercellular communication networks—particularly between cytotoxic cells and their regulators—altered expression of genes implicated in disease progression (OLR1, SERPINB2, COPS9) and viral persistence control (NEAT1, NAF1). This work provides an initial single-cell transcriptional atlas characterizing early immune responses to HIV-1 sub-subtypes A6 and CRF63_02A6, the predominant drivers of the HIV epidemic across the FSU region.

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.

Against the backdrop of active efforts to combat HCV worldwide with the help of DAAs, knowledge of the genetic diversity of HCV in the general population and in groups most at risk of infection is becoming increasingly important. The aim of this study was to characterize the molecular genetic diversity of HCV among individuals with HIV in Armenia, Kyrgyzstan and the Krasnoyarsk Krai region of Russia. The study included residents of Armenia (n = 73), Kyrgyzstan (n = 180) and the Krasnoyarsk Territory (n = 141) with HIV/HCV co-infection who were under observation at AIDS centers in these countries, collected between 2021 and 2023. The Core/E1 gene fragments obtained were analyzed using the maximum likelihood method to create a phylogenetic tree. HCV subtype 3a was dominant in Armenia (56.2%) and Kyrgyzstan (51.4%). The circulation of HCV subtype 4a was detected for the first time in Armenia, while the spread of HCV genotype 2, represented by three different subtypes, was documented in Kyrgyzstan. The genetic diversity of HCV in Krasnoyarsk Krai is consistent with the findings of previous Russian studies. Phylogenetic analysis revealed the formation of HCV clusters with a high level of bootstrap support, suggesting shared transmission routes, predominantly among PWID. This suggests that there are common routes of HCV transmission between and within countries.

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.

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 ad-dress 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 parame-ter—signal distribution entropy—to quantify the uniformity of compound distribution within cells. For statistical analysis, we developed FluoSta v1.0, a software tool that auto-mates 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.

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.

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.