HIV-1 Genome Research Articles

DNAzyme-Triggered Equilibrium Transfer with Self-Activated CRISPR-Cas12a Biosensor Enables One-Pot Diagnosis of Nucleic Acids.

Integrating recombinase-polymerase amplification (RPA) with CRISPR-Cas12a holds significant potential to simplify and improve nucleic acid diagnostic procedures. However, current strategies face limitations, such as complexity, reduced efficiency, and potential compromises in Cas12a activity. In response, we developed a DNAzyme-triggered equilibrium transfer with a self-activated CRISPR-Cas12a biosensor (DESCRIBER) for integrated nucleic acid detection. This platform features varying balance points to minimize interference between RPA and Cas12a in one pot and maximize their activity at different stages. Initially, the reaction focused on RPA, while Cas12a was silenced by circular-crRNA (C-crRNA). Then, DNAzyme, the activator, was generated during the RPA process, which linearizes C-crRNA to activate Cas12a and transfer the equilibrium toward signal readout. Meanwhile, activated Cas12a can further linearize C-crRNA to promote self-activation and accelerate equilibrium transfer. According to this principle, highly sensitive detection of the HIV-1 genome, as low as 500 CPs/mL, was achieved within 1 h while maintaining universality in detecting common subtypes and specificity against opportunistic infectious pathogens. Compared with qRT-PCR, it also exhibited good accuracy in detecting 35 spiked samples. Overall, we believe that the proposed strategy will enhance existing CRISPR systems to promote their practical applications in clinical diagnosis.

P-490. Differentiation of Latent Versus Active HIV Infection at the Single Cell Level Using RT-ddPCR

Abstract Background With successful treatment of HIV, viral loads can drop to levels undetectable by regular qPCR diagnostic methods. However, the virus remains latent in cellular reservoirs . These reservoir cells are rare, and their quantification is a critical parameter to assess future HIV treatments. Digital PCR (dPCR) technology bypasses the limitation inherent to regular qPCR, and can quantify to some extent cells with latent HIV infection within a cell population. Methods Here we describe a protocol relying on dPCR that allows differentiation between active and latent infection at the single cell level. We used a digital droplet setting (ddPCR) that allows for reverse transcription (RT) within the droplet. The assay is multiplexed to detect both HIV proviral DNA as well as spliced mRNA indicative of HIV active replication. Briefly, single cell are isolated into droplet containing RT-ddPCR reaction, including the Bio-Rad Supermix One-Step RT ddPCR Advanced Kit and primers/probe sets targeting HIV genome gag in the FAMlow channel and the spliced 2kb class mRNA in the FAMhigh channel. After the RT-PCR reaction, the droplet fluorescence intensity was measured using a Bio-Rad Q200 droplet reader. Results We successfully evaluated HIV activation in cells lines with (8E5) or without HIV (CEM-T4) infection, as well as volunteer Peripheral Blood Mononuclear Cells (PBMC). We first determine the fluorescence bandwith for each target using DNA/RNA purified from cell lysate, identifying the HIV genomic DNA signal (FAMlow) between 2500 and 3500 MFI and the active replication signal (FAMhigh) between 4000 and 5000 MFI. We then repeated the RT-ddPCR reaction, this time using cells isolated in an excess of droplets (2000 cells for 15,000 droplet per reaction). Conclusion This Single Cell RT-ddPCR protocol allows for identification and quantification of HIV infected cells and assess their individual level of activation/latency. In the future, the multiplexing approach could be used to not only assess HIV activation, but also to identify cellular transcripts specific to latency or active replication. Disclosures All Authors: No reported disclosures

P-492. Quantification of HIV Shedding in Viremic Patients

Abstract Background Identifying pockets of undiagnosed HIV is a critical step in the U.S. Ending the HIV Epidemic (EHE). Wastewater-based epidemiology represents a novel way to build agnostic population-level data about disease distribution. We detected HIV virus in two sewersheds in Northern California. We are now conducting a study to measure how much HIV virus is shed into urine and stool, to better estimate cases represented by wastewater detection. HIV-1 RNA Detected in Concentrated Urine Methods We are recruiting participants from HIV clinics in San Mateo and Santa Clara Counties. Patients must be 18 years or older with HIV-1 viral load of >10,000 copies/ml at the time of recruitment. Subjects are asked to provide 5 weekly self-collected blood (Tasso), urine, and stool samples. The plasma is analyzed by established HIV-1 RNA quantitative PCR techniques. Nucleic acids from urine, concentrated (centrifuged) urine, and stool are suspended in DNA/RNA Shield, then extracted using the ZymoBIOMICS DNA/RNA Miniprep Kit (ZymoBIOMICS #R2002). HIV genome-specific primers target the Long Terminal Repeat (LTR) region of the HIV-1 genome. Droplets for digital droplet PCR are generated using the AutoDG Automated Droplet Generator. Reverse transcriptase PCR, which quantifies RNA and DNA combined, and direct PCR, which quantifies DNA, is then carried out with the Mastercycler Pro. Thresholding is performed using QX Manager Software. HIV-1 RNA Detected in Stool Results Two patients have been enrolled to date. Patient 1 was started on antiretroviral therapy after the first sample was obtained; Patient 2 was started on antiretroviral therapy 10 days before the first sample was collected. HIV-1 RNA was found in 87.5% (7/8) of concentrated urine samples, including 85.7% (6/7) of samples obtained while on ART (Table 1). Among stool samples, 25.0% (2/8) yielded HIV RNA, but only 14.3% (1/7) of those obtained on ART (Table 2). Nucleic acids were not detected in unconcentrated urine. Plasma viral load testing is pending, so only the initial clinical HIV-1 viral loads at enrollment are shown. Conclusion HIV-1 RNA is present in concentrated urine but also in stool samples of viremic patients, even up to five weeks after initiation of ART. When the study is complete, our results will bolster how to interpret HIV-1 detected in wastewater. Disclosures All Authors: No reported disclosures

Reactivation of latent HIV-1 by the glucocorticoid receptor modulator AZD9567.

One key determinant of HIV-1 latency reversal is the activation of the viral long terminal repeat (LTR) by cellular transcription factors such as NF-κB and AP-1. Interestingly, the activity of these two transcription factors can be modulated by glucocorticoid receptors (GRs). Furthermore, the HIV-1 genome contains multiple binding sites for GRs. We therefore hypothesized that glucocorticoids and other GR modulators may influence HIV-1 latency and reactivation. To investigate how GR signaling affects latent HIV-1 reservoirs, we assembled a representative panel of GR modulators including natural steroidal agonists, selective and non-selective GR modulators, and clinically approved GR-modulating drugs. The effects of these compounds on HIV-1 reactivation were assessed using latently HIV-1-infected cell lines and primary cells, as well as reporter assays that monitored GR and LTR activities. We found that AZD9567 (Mizacorat), a non-steroidal partial GR agonist, reactivates latent HIV-1 in both lymphoid and myeloid cell lines and primary CD4+ T cells. Conversely, the GR antagonist mifepristone suppresses HIV-1 LTR-driven gene expression. Mechanistic analyses revealed that AZD9567-mediated reactivation partially depends on both GR and AP-1 binding sites in the LTR. In summary, we, here, identify the GR modulator AZD9567 as novel latency-reversing agent that activates LTR-driven gene expression, which may aid in advancing current shock-and-kill approaches in the treatment of HIV-1 infection.IMPORTANCELatently infected cells of people living with HIV are constantly exposed to fluctuating levels of glucocorticoid hormones such as cortisol. In addition, many HIV-infected individuals regularly take corticosteroids as anti-inflammatory drugs. Although corticosteroids are known to affect the activity of the viral long terminal repeat (LTR) promoter and influence ongoing HIV-1 replication, relatively little is known about the effect of corticosteroid hormones and other glucocorticoid receptor (GR) modulators on latent HIV-1. By systematically comparing natural and synthetic GR modulators, we, here, identify a first first-in-class, oral, partial GR agonist that reactivates latent HIV-1 from different cell types. This drug, AZD9567, was previously tested in clinical trials for rheumatoid arthritis. Mutational analyses shed light on the underlying mode of action and revealed transcription factor binding sites in the HIV-1 LTR that determine responsiveness to AZD9567.

Brain RNA profiling highlights multiple disease pathways in persons with HAND: uncovering determinants of biotype diversity.

To discover microRNA (miRNA)-RNA transcript interactions dysregulated in brains from persons with HIV-associated neurocognitive disorder (HAND), we investigated RNA expression using machine learning tools. Brain-derived host RNA transcript and miRNA expression was examined from persons with or without HAND using bioinformatics platforms. By combining next generation sequencing, droplet digital (dd)PCR quantitation of HIV-1 genomes, with bioinformatics and statistical tools, we investigated differential RNA expression in frontal cortex from persons without HIV (HIV[-]), with HIV without brain disease (HIV[+]), with HIV-associated neurocognitive disorder (HAND), or HAND with encephalitis (HIVE). Expression levels for 147 transcripts and 43 miRNAs showed a minimum 4-fold difference between clinical groups with a predominance of antiviral (Type I interferon) signaling-, neural cell maintenance-, and neurodevelopmental disorder-related genes that was validated by gene ontology and molecular pathway inferences. Scale of signal-to-noise ratio (SSNR) and biweight midcorrelation (bicor) analyses identified 14 miRNAs and 45 RNA transcripts, which were highly correlated and differentially expressed (p ≤ 0.05). Machine learning applications compared regression models predicated on HIV-1 DNA, or RNA viral quantities that disclosed miR-4683 and miR-154-5p were dominant variables associated with differential expression of host RNAs. These miRNAs were also associated with antiviral-, cell maintenance-, and neurodevelopmental disorder-related genes. Antiviral as well as neurodevelopmental disorder-related pathways in brain were associated with HAND, based on correlated RNA transcripts and miRNAs. Integrated molecular methods with machine learning offer insights into disease mechanisms, underpinning brain-related biotypes among persons with HIV that could direct clinical care.

HIV-1 drug resistance and genetic transmission networks among patients with sexually transmitted HIV in Ningxia, China.

Over the past decade, sexual transmission has become a dominant source of new HIV-1 infection in China. However, very few studies have been conducted to characterize the two sexual transmissions, homosexual and heterosexual transmission. This study was conducted to better understand the relationship between genotypes, drug resistance, and molecular transmission networks in two groups of sexually transmitted HIV-1 in Ningxia, China. Plasma samples were collected from sexually transmitted HIV/AIDS patients in Ningxia between 2020 and 2021 for RNA extraction followed by HIV-1 genome sequencing, genotype and drug resistance analyses. The TN93 model in HyPhy2.2.4 with 1.25% as the threshold, was used to calculate the gene distance, and Cytoscape3.7.0 was used to generate a visual molecular transmission network. A total of 269 samples were successfully sequenced, and 10 HIV-1 subtypes were detected. The two most common subtypes were CRF07_BC and CRF01_AE. All 10 subtypes were detected in heterosexually transmitted patients, and 7 subtypes were found in homosexually transmitted patients who were exclusively men sex with men (MSM). The drug resistance rates of heterosexual individuals and MSMs were 45.34 and 33.33%, respectively. Sequences from 120 patients entered the molecular transmission network, forming 35 clusters. The clustering rate for MSM (52.78%) was higher than that of heterosexual individuals (39.13%). Some MSM and HSTs were involved in the same cluster and might act as bridges for transmission between the two populations. Our data showed that heterosexually transmitted HIV-1 was more likely to be a drug-resistant virus, whereas MSM was more likely to contract viruses through network connection. It is strongly recommended that resistance testing be conducted before ART to improve effective treatment and reduce the spread of resistant viruses. Molecular networks can help to identify transmission clusters and provide more precise interventions.

Post-Integrational DNA Repair of HIV-1 Is Associated with the Activation of DNA-PK and ATM Cellular Protein Kinases and Phosphorylation of Their Targets

Integration of the DNA copy of the HIV-1 genome into the cellular genome results in series of damages, the repair of which is critical for successful viral replication. We have previously demonstrated that the ATM and DNA-PK kinases, normally responsible for repairing double-strand breaks in the cellular DNA, are required to initiate HIV-1 post-integration repair, even though integration does not result in double-strand DNA breaks. In this study, we analyzed changes in the phosphorylation status of ATM (pSer1981), DNA-PK (pSer2056) and their related kinase ATR (pSer428), as well as their targets: Chk1 (pSer345), Chk2 (pThr68), H2AX (pSer139) and p53 (pSer15) during HIV-1 post-integration repair. We have shown that ATM and DNA-PK, but not ATR, undergo autophosphorylation during postintegration DNA repair and phosphorylate their target proteins Chk2 and H2AX. These data indicate common signaling mechanisms between double-strand DNA break repair and postintegration repair of HIV-1.

HIV-SEQ REVEALS GLOBAL HOST GENE EXPRESSION DIFFERENCES BETWEEN HIV-TRANSCRIBING CELLS FROM VIREMIC AND SUPPRESSED PEOPLE WITH HIV.

"Active" reservoir cells transcribing HIV can perpetuate chronic inflammation in virally suppressed people with HIV (PWH) and likely contribute to viral rebound after antiretroviral therapy (ART) interruption, so they represent an important target for new therapies. These cells, however, are difficult to study using single-cell RNA-seq (scRNA-seq) due to their low frequency and low levels of HIV transcripts, which are usually not polyadenylated. Here, we developed "HIV-seq" to enable more efficient capture of HIV transcripts - including non-polyadenylated ones - for scRNA-seq analysis of cells from PWH. By spiking in a set of custom-designed capture sequences targeting conserved regions of the HIV genome during scRNA-seq, we increased our ability to find HIV RNA+ cells from PWH by up to 44%. Implementing HIV-seq in conjunction with surface phenotyping by CITE-seq on paired blood specimens from PWH before vs. after ART suppression, we found that HIV RNA+ cells were enriched among T effector memory (Tem) cells during both viremia and ART suppression, but exhibited a cytotoxic signature during viremia only. By contrast, HIV RNA+ cells from the ART-suppressed timepoints exhibited a distinct anti-inflammatory signature involving elevated TGF-β and diminished IFN signaling. Overall, these findings demonstrate that active reservoir cells exhibit transcriptional features distinct from HIV RNA+ cells during viremia, and underscore HIV-seq as a useful tool to better understand the mechanisms by which HIV-transcribing cells can persist during ART.

The features of Tat protein of human immunodeficiency virus type 1 (Retroviridae: Lentivirus: Lentivirus humimdef1) non-A6 variants, characteristic for the Russian Federation.

Tat protein is a trans-activator of HIV-1 genome transcription, with additional functions including the ability to induce the chronic inflammatory process. Natural amino acid polymorphisms in Tat may affect its functional properties and the course of HIV infection. The aim of this work is to analyze the marks of Tat consensus sequences in non-A6 HIV-1 variants characteristic of the Russian Federation, as well as study natural polymorphisms in Tat CRF63_02A6 and subtype B variants circulating in Russia. The whole-genome nucleotide sequences of HIV-1 CRF63_02A6, CRF03_A6B, as well as subtype B and CRF02_AG circulating in Russia were used. The reference group was formed based on the sequences of subtype B variants circulating in different countries. Preferentially, the sequences were downloaded from the international database Los Alamos. CRF63_02A6 consensus sequence contained the highest number of amino acid substitutions, 31, and had no helix at positions 30‒33 in the secondary structure; however, this did not change its predicted tertiary structure. CRF03_A6B consensus sequence contained a stop codon at position 87. The polymorphisms in subtype B variants circulating in our country and in CRF63_02A6 variants were identified. Consensus sequences of Tat protein in non-A6 variants typical for the Russian Federation were obtained and their features were determined. R78G, located in the functionally significant motif, and C31S, the functionally significant substitution, were significantly more frequent in subtype B variants circulating in Russia and in CRF63_02A6 variants than in the reference group, respectively. A limitation of this study is the small sample of sequences.

Disruption of LEDGF/p75-directed integration derepresses antisense transcription of the HIV-1 genome.

Disruption of HIV-1 Integrase (IN) interactions with the host-factor Lens Epithelium-Derived Growth Factor (LEDGF)/p75 leads to decreased, random integration, increased latent infection, and described here, accumulation of HIV-1 antisense RNA (asRNA). asRNA increase was observed following interruptions of IN-LEDGF/p75 interactions either through pharmacologic perturbations of IN-LEDGF/p75 by treatment with allosteric HIV-1 integrase inhibitors (ALLINIs) or in cell lines with LEDGF genetic knockout. Additionally, by impairing Tat-dependent HIV transcription, asRNA abundance markedly increases. Illumina sequencing characterization of asRNA transcripts in primary T cells infected in the presence of ALLINIs showed that most initiate from within the HIV-1. Overall, loss of IN-LEDGF/p75 interactions increase asRNA abundance. Understanding the relationship between ALLINIs, integration sites, asRNA, and latency could aid in future therapeutic strategies.

Decoding the biogenesis of HIV-induced CPSF6 puncta and their fusion with the nuclear speckle.

Viruses rely on host cellular machinery for replication. After entering the nucleus, the HIV genome accumulates in nuclear niches where it undergoes reverse transcription and integrates into neighboring chromatin, promoting high transcription rates and new virus progeny. Despite anti-retroviral treatment, viral genomes can persist in these nuclear niches and reactivate if treatment is interrupted, likely contributing to the formation of viral reservoirs. The post-nuclear entry dynamics of HIV remain unclear, and understanding these steps is critical for revealing how viral reservoirs are established. In this study, we elucidate the formation of HIV-induced CPSF6 puncta and the domains of CPSF6 essential for this process. We also explore the roles of nuclear speckle scaffold factors, SON and SRRM2, in the biogenesis of these puncta. Through genetic manipulation and depletion experiments, we demonstrate the key role of the intrinsically disordered region of SRRM2 in enlarging nuclear speckles in the presence of the HIV capsid. We identify the FG domain of CPSF6 as essential for both puncta formation and binding to the viral core, which serves as the scaffold for CPSF6 puncta. While the low-complexity regions (LCRs) modulate CPSF6 binding to the viral capsid, they do not contribute to puncta formation, nor do the disordered mixed charge domains (MCDs) of CPSF6. These results demonstrate how HIV evolved to hijack host nuclear factors, enabling its persistence in the host. Of note, this study provides new insights into the underlying interactions between host factors and viral components, advancing our understanding of HIV nuclear dynamics and offering potential therapeutic targets for preventing viral persistence.

PP2.1 – 00175 HIV genome derived from the brain microglia isolated from PWH on ART

PP2.1 – 00175 HIV genome derived from the brain microglia isolated from PWH on ART

1.3 – 00084 Persistence of HIV genomes in bacteria-specific CD4+ T cells during ART

1.3 – 00084 Persistence of HIV genomes in bacteria-specific CD4+ T cells during ART

Defective HIV proviruses: possible involvement in the HIV infection pathogenesis.

This review article analyzes information obtained from a literature search on defective HIV genomes (HIV-1, Human Immunodeficiency Virus, Lentivirus, Orthoretrovirinae, Retroviridae). It discusses the origins of defective HIV genomes, their potential for transcription and translation, and the role of defective RNA and proteins in stimulating both innate and adaptive immunity. The article also explores their contribution to HIV pathogenesis, immune system hyperactivation despite successful antiretroviral therapy (ART), and the evolutionary processes in HIV proviral populations under ART. Additionally, it addresses challenges in reservoir elimination and HIV eradication that arise from the existence of defective HIV viruses.

Minimally Modified HIV-1 Infection of Macaques: Development, Utility, and Limitations of Current Models.

Nonhuman primate (NHP) studies that utilize simian immunodeficiency virus (SIV) to model human immunodeficiency virus (HIV-1) infection have proven to be powerful, highly informative research tools. However, there are substantial differences between SIV and HIV-1. Accordingly, there are numerous research questions for which SIV-based models are not well suited, including studies of certain aspects of basic HIV-1 biology, and pre-clinical evaluations of many proposed HIV-1 treatment, prevention, and vaccination strategies. To overcome these limitations of NHP models of HIV-1 infection, several groups have pursued the derivation of a minimally modified HIV-1 (mmHIV-1) capable of establishing pathogenic infection in macaques that authentically recapitulates key features of HIV-1 in humans. These efforts have focused on three complementary objectives: (1) engineering HIV-1 to circumvent species-specific cellular restriction factors that otherwise potently inhibit HIV-1 in macaques, (2) introduction of a C chemokine receptor type 5 (CCR5)-tropic envelope, ideally that can efficiently engage macaque CD4, and (3) correction of gene expression defects inadvertently introduced during viral genome manipulations. While some progress has been made toward development of mmHIV-1 variants for use in each of the three macaque species (pigtail, cynomolgus, and rhesus), model development progress has been most promising in pigtail macaques (PTMs), which do not express an HIV-1-restricting tripartite motif-containing protein 5 α (TRIM5α). In our work, we have derived a CCR5-tropic mmHIV-1 clone designated stHIV-A19 that comprises 94% HIV-1 genome sequence and replicates to high acute-phase titers in PTMs. In animals treated with a cell-depleting CD8α antibody at the time of infection, stHIV-A19 maintains chronically elevated plasma viral loads with progressive CD4+ T-cell loss and the development of acquired immune-deficiency syndrome (AIDS)-defining clinical endpoints. However, in the absence of CD8α+ cell depletion, no mmHIV-1 model has yet displayed high levels of chronic viremia or AIDS-like pathogenesis. Here, we review mmHIV-1 development approaches, the phenotypes, features, limitations, and potential utility of currently available mmHIV-1s, and propose future directions to further advance these models.

Comparison of short-read and long-read next-generation sequencing technologies for determining HIV-1 drug resistance.

Accurate HIV-1 genome sequencing is necessary to identify drug resistance mutations (DRMs) in people with HIV-1 (PWH). Next-generation-sequencing (NGS) allows the detection of minor variants and is now available in many laboratories. Our study aimed to compare two NGS approaches, a "short read" sequencing protocol using DeepChek® Whole Genome HIV-1 Assay on Illumina, and a "long read" sequencing protocol of HIV-1 pol and env single-molecule real-time sequencing (SMRT) on Pacific Biosciences (PacBio). We analyzed 16 plasma samples and 13 cellular samples from PWH. HIV-1 whole genome was amplified into five amplicons using DeepChek® Whole Genome HIV-1 Assay and sequenced on an iSeq. 100. In parallel, HIV-1 pol and env genes were separately amplified and sequenced using PacBio SMRT system with the circular consensus sequencing mode on a Sequel IIe. Concordance rates for determining DRMs with both approaches varied depending on the HIV-1 region, with higher concordance in the integrase region compared to the reverse transcriptase and protease regions. DeepChek® Whole Genome HIV-1 Assay exhibited better sensitivity in HIV-1 RNA sequencing of plasmas with lower viral loads. In cell HIV-1 DNA sequencing, the DeepChek® Whole Genome HIV-1 Assay performed better in pol and env sequencing but detected more APOBEC-induced DRMs, which can represent defective proviruses. Our findings indicate that both DeepChek® Whole Genome HIV-1 Assay and PacBio SMRT sequencing exhibit good performance for subtype determination, detection, and quantification of DRMs of the HIV-1 genome. However, some discrepancies were found in cellular samples, highlighting the challenges of interpreting HIV-1 DNA DRMs.

Intact HIV reservoir in monocytes is associated with cognitive function in virally suppressed women with HIV.

Monocytes are susceptible to HIV infection, form HIV reservoirs, and contribute to central nervous system complications (e.g., cognitive impairment) in virally suppressed women with HIV(vsWWH). However, it remains unknown if the quality and/or quantity of the monocyte reservoir contributes to cognition in vsWWH. 62 vsWWH(mean age=56.1, SD=7.1; 93% Black, non-Hispanic; all HIV RNA <250 copies/mL) completed a cognitive test battery, blood draw, and whole blood immunophenotyping. Monocytes and CD4 T cells were isolated from a subset of 53 participants and the HIV reservoir was assessed using cell specific Intact Proviral DNA Assays(IPDA). Demographically-adjusted z-scores were calculated for each outcome using data from participants without HIV in the Women's Interagency HIV Study. Cognitive outcomes of interest included domain-specific and global z-scores. Thirty-Eight percent of vsWWH had detectable intact HIV genomes in monocytes(median=21.5 copies/million). Higher levels of intact HIV genomes per million monocytes were associated with poorer verbal memory(delayed recall: r=0.55, P=0.01; recognition: r=0.46, P=0.04), fine motor skills(r=0.50, P=0.03), and global function(r=0.47, P=0.04). Higher levels of intact HIV genomes in monocytes were associated with percent intermediate monocytes(r=0.60, P=0.008), and the ratio of intact per intermediate monocyte was associated with worse memory(r=-0.59, P=0.008). There were no associations between CD4 reservoir and cognition. The number of intact HIV genomes per million monocytes were related to poorer cognition and the percentage of intermediate monocytes. These findings suggest that the presence of HIV genomes in general do not relate to cognitive complications, but intact, and therefore potentially replication-competent HIV, may contribute to cognitive complications in vsWWH.

Identification of antibody targets associated with lower HIV viral load and viremic control.

High HIV viral loads (VL) are associated with increased morbidity, mortality, and on-going transmission. HIV controllers maintain low VLs in the absence of antiretroviral therapy (ART). We previously used a massively multiplexed antibody profiling assay (VirScan) to compare antibody profiles in HIV controllers and persons living with HIV (PWH) who were virally suppressed on ART. In this report, we used VirScan to evaluate whether antibody reactivity to specific HIV targets and broad reactivity across the HIV genome was associated with VL and controller status 1-2 years after infection. Samples were obtained from participants who acquired HIV infection in a community-randomized trial in Africa that evaluated an integrated strategy for HIV prevention (HPTN 071 PopART). Controller status was determined using VL and antiretroviral (ARV) drug data obtained at the seroconversion visit and 1 year later. Viremic controllers had VLs <2,000 copies/mL at both visits; non-controllers had VLs >2,000 copies/mL at both visits. Both groups had no ARV drugs detected at either visit. VirScan testing was performed at the second HIV-positive visit (1-2 years after HIV infection). The study cohort included 13 viremic controllers and 64 non-controllers. We identified ten clusters of homologous peptides that had high levels of antibody reactivity (three in gag, three in env, two in integrase, one in protease, and one in vpu). Reactivity to 43 peptides (eight unique epitopes) in six of these clusters was associated with lower VL; reactivity to six of the eight epitopes was associated with HIV controller status. Higher aggregate antibody reactivity across the eight epitopes (more epitopes targeted, higher mean reactivity across all epitopes) and across the HIV genome was also associated with lower VL and controller status. We identified HIV antibody targets associated with lower VL and HIV controller status 1-2 years after infection. Robust aggregate responses to these targets and broad antibody reactivity across the HIV genome were also associated with lower VL and controller status. These findings provide novel insights into the relationship between humoral immunity and viral containment that could help inform the design of antibody-based approaches for reducing HIV VL.

A multiplexed real‐time PCR assay for simultaneous quantification of human immunodeficiency virus and Hepatitis B virus for low‐and‐middle‐ income countries

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16–74.3] for HBV and 82 % [78.09–85.90] for HIV and an overall specificity of 100 % [94.95–100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26–88.73] and >1000 IU/mL for HBV was 100 % [95.51–100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.

Galectin-9 Levels as a Potential Predictor of Intact HIV Reservoir Decay.

During antiretroviral therapy (ART), the HIV reservoir exhibits variability as cells with intact genomes decay faster than those with defective genomes, especially in the first years of therapy. The host factors influencing this decay are yet to be characterized. Observational study in 74 PWH on ART, of whom 70 (94.6%) were male. We used the intact proviral DNA assay to measure intact proviruses and Luminex immunoassay to measure 32 inflammatory cytokines in plasma. Linear spline models, with a knot at seven years, evaluated the impact of baseline cytokine levels and their trajectories on intact HIV kinetics over these years. Baseline Gal-9 was the most predictive marker for intact HIV kinetics, with lower Gal-9 predicting faster decay over the subsequent seven years. For each 10-fold decrease in Gal-9 at baseline, there was a mean 45% (95%CI 14%-84%) greater decay of intact HIV genomes per year. Conversely, higher baseline ITAC, IL-17, and MIP-1α predicted faster intact HIV decreases. Longitudinal changes in MIP-3α and IL-6 levels strongly associated with intact HIV kinetics, with a 10-fold increase in MIP-3α and a 10-fold decrease in IL-6 associated with a a 9.5% and 10% faster decay of intact HIV genomes per year, respectively. The pronounced association between baseline Gal-9 levels and subsequent intact HIV decay suggests that strategies reducing Gal-9 levels could accelerate reservoir decay. Additionally, the correlations of MIP-3α and IL-6 with HIV kinetics indicate a broader cytokine-mediated regulatory network, hinting at multi-targeted interventions that could modulate HIV reservoir dynamics.