Introduction: The intact proviral DNA assay (IPDA), widely used for HIV reservoir quantification, is a key tool in cure research. This droplet digital PCR assay simultaneously targets two HIV regions, psi and the rev response element in env, allowing intact proviruses to be discriminated from defective ones. Due to naturally-occurring HIV polymorphism however, signal failure in one or both targets occurs in up to 30% of samples, necessitating custom primer/probe design and greatly increasing the assay's resource burden. We systematically investigated IPDA tolerance of HIV polymorphism with the goal of improving the assay.

Methods: We applied the IPDA to 140 individuals with HIV receiving ART. We sequenced the IPDA target regions from plasma where available or from on-ART proviral DNA using Sanger (92%), Illumina MiSeq (6%), or both (2%). IPDA signal amplitudes were interpreted alongside the autologous sequences, allowing us to determine the effects of individual HIV polymorphisms on assay performance.

Results: Full signal failure occurred in 27 (19%) samples. Partial failure, where the IPDA detected only a subset of all within-host HIV species, occurred in an additional 20 (14%). Polymorphisms that caused failure were a deletion of the A at base 19 of the forward psi primer (observed in 8% samples), and G→A mutations at bases 7 and/or 13 of the env probe (observed in 3.8% and 10.5% of samples respectively). In general, probe mismatches reduced signal amplitude in a position-dependent manner, while primer mismatches were broadly tolerated. Mismatches at base 2 of the env probe, observed in 11.5% of samples, reduced amplitude most severely though gating was still possible in some cases.

Conclusions: These results can help us refine current IPDA primers/probes and design alternative ("rescue") oligos. Partial assay failures due to within-host polymorphism remain a challenge with the IPDA, because these are impossible to detect without sequencing.

Background. The primary barrier to an HIV cure is the latent viral reservoir (LVR), a product of HIV’s integration into the DNA of long-lived immune cells, predominantly resting CD4+ T cells. The Intact Proviral DNA Assay was designed based on HIV subtype B (IPDA-B) as a scalable method for LVR quantification. We adapted the IPDA-B for the sequence diversity among 23 people with HIV (PWH) subtypes A1, D, and recombinants in Uganda (IPDA-A1D). Here, we evaluated this assay’s performance across a broader Ugandan cohort and made additional design modifications to address assay failures.

Methods. The IPDA-A1D was performed on banked PBMC samples from n=64 PWH in Rakai, Uganda. CD4+ T cells were negatively selected, genomic DNA isolated, and duplex droplet digital PCR was performed. Nucleotide diversity in IPDA-A1D primer/probe regions was evaluated using HIV proviral sequence data. Published IPDA-A1D primers/probes were refined and applied to samples for which amplification failed in one or both regions.

Results. The IPDA-A1D successfully quantified the LVR in 68.8% of participants, with an additional 7.8% having indeterminate results. The failure rate was 23.4%, with 41.2% of failures occurring in the psi region, 52.9% in the env region, and 5.9% in both regions. Subsequent primer and probe modifications rescued 91.7% of failures, raising the success rate to 85.9%. In general, fluorescence amplitude decreased with higher numbers of degenerate bases within the probe or with more probe variants included in a reaction, particularly in env, where this strategy rescued failures but decreased signal amplitude, making gating difficult.

Significance. Adapting the IPDA for global populations with diverse HIV subtypes requires a delicate balance between sequence diversity and fluorescence intensity. As HIV subtypes differ in viral pathogenesis, genetics, and reservoir sizes, subtype-specific assays for accurate LVR quantification are needed to evaluate reservoir outcomes in African populations.

HIV-1 integration into the host genome is a critical step in infection that influences viral gene expression, latency, and long-term persistence during antiretroviral therapy. Although integrase inhibitors are highly effective at suppressing viral replication, how prolonged exposure to these drugs influences HIV-1 integration targeting over time remains poorly defined. Here, we combined longitudinal integration-site profiling with integrase genetic analysis, genome-wide proviral sequencing, and analysis of recombinant viruses carrying fixed integrase variants to examine the effects of sustained dolutegravir (DTG) and raltegravir (RAL) exposure on HIV-1 integration targeting using NL4-3 viruses carrying subtype A-, B-, or D-derived integrase sequences. By comparing integration patterns over time to matched no-drug controls, we found that both DTG and RAL drive progressive, structured remodeling of integration-site profiles that exceeds long-term culture-associated drift. These changes reflected coordinated reweighting across overlapping gene-associated, regulatory, and structural chromatin environments rather than enrichment of individual genomic features. While all integrase variants exhibited drug-associated remodeling, the magnitude and kinetics of these changes differed by variant and drug, with RAL producing more rapid and heterogeneous effects than DTG. Notably, altered integration targeting was detectable in viruses carrying fixed integrase resistance mutations, indicating that integration-site remodeling can arise directly from integrase sequence variation and is not strictly coupled to classical resistance pathways or broader genome-wide proviral evolution. Together, these findings demonstrate that integrase inhibitor pressure reshapes HIV-1 integration targeting in an integrase variant–dependent manner, with potential implications for viral reservoir composition and long-term persistence during therapy.

Introduction
In people with HIV (PWH) on antiretroviral therapy (ART), HIV persists in long-lived CD4+ T-cells, constituting the main obstacle to a cure. We recently reported that a large fraction (61%) of inducible, translation competent HIV genomes in PWH on ART present genetic defects in the highly structured Ψ RNA packaging signal. Because structured viral RNA can be sensed by the innate immune sensors RIG-I and MDA5, leading to cell death, we hypothesized that innate immune pressure drives the selection of defective proviruses during ART.

Methodology
To determine if cells carrying genetically intact Ψ-element are selectively killed upon reactivation, CD4+ T-cells from 3 PWH on ART were stimulated with PMA/ionomycin for 24h with or without the apoptosis inhibitor Q-VD-Oph. The genetic integrity of the Ψ-element was assessed by sequencing HIV genomes in sorted p24+ cells. Using site-directed mutagenesis or cloning of proviral sequences from PWH, we generated infectious molecular clones carrying partial or complete deletion of the Ψ-region. After transfection, the activation of RIG-I and MDA5 pathways was assessed by measuring IFN-β and IFIT-1 by RT-qPCR.

Results
Primary CD4+ T-cells constitutively expressed RIG-I and MDA5, with increased expression following TCR stimulation and IFN-α treatment (3- and up to 60-fold, respectively). Analysis of 32 proviral sequences from single sorted p24+ cells showed no enrichment of intact genomes upon Q-VD-Oph treatment, challenging our original hypothesis. In contrast, transfection of infectious molecular clones carrying genetically intact Ψ-element induced higher IFN-β and IFIT-1 expression than Ψ-defective clones, suggesting that a specific motif engages RIG-I and MDA5.

Conclusion
Our results suggest a mechanism by which innate immune sensors actively contribute to the negative selection of genetically intact HIV genomes during ART. Whether the induction of RIG-I and MDA5 can be leveraged to accelerate the depletion of intact HIV genomes remains to be determined.

Antiretroviral therapy (ART) suppresses Human Immunodeficiency Virus-1 (HIV) replication to undetectable levels in people living with HIV (PLWH), successfully reducing mortality rates. However, ART is not curative due to the establishment of the HIV reservoir that persists despite prolonged ART suppression. It has been established that long-lived memory T cells represent the most important HIV reservoir population which is predominantly composed of CD4+ T cell clones that are established early after infection and increase under ART treatment. While factors such as integration site effects have been proposed to induce clonal expansion of HIV-infected T cells, our data points to cognate dendritic cell:T cell interactions as the main driver of clonal expansion of latent T cell subsets through T cell receptor (TCR)-dependent signaling and cytokines that support a pro-survival state in these cells. Depending on the nature of the TCR stimulus, latent T cell proliferation can occur in the absence of virus reactivation. However, a remaining gap in knowledge is how antigen stimulation regulates opposing biological processes of (i) proliferation leading to clonal expansion or (ii) viral production leading to cell death. Here, we utilized a dual-fluorescent HIV latency reporter and antigen-specific human CD4+ T cell clones to modulate TCR signaling magnitudes using a panel of altered peptide ligands of various avidities. We directly examined the relationship between TCR signals and proliferative responses by latent T cells. Our preliminary data suggests that low avidity antigen stimulation drives proliferation without viral reactivation in latent T cell subsets while high avidity stimulation promotes virus reactivation and cell death. Our data argue that a critical balance between stimulatory and inhibitory pathways dictate which T cell subsets clonally expand or undergo apoptosis under ART suppression. These studies have implications on stimulatory signals that can be therapeutically targeted to reduce the HIV reservoir size in PLWH.

Human Immunodeficiency Virus Type 1 (HIV-1) persists in people living with HIV-1 (PLWH) on antiretroviral treatment (ART) due to a latently infected immune cell population termed the latent reservoir. Most latent reservoir research focuses on subtype B HIV-1, with limited studies on the non-subtype B strains prevalent in lower-income countries. We previously characterized the Major Histocompatibility Complex Class I (MHC-I) and CD4 downregulation ability of non-Subtype B Nef proteins from ART-suppressed PLWH in Uganda and found a correlation between the decay rate of the latent reservoir and Nef-mediated MHC-I downregulation, but not CD4 downregulation. However, it remains unknown if other Nef functions can affect latent reservoir dynamics in PLWH. Of interest is SERINC5, a host restriction factor that reduces progeny virion infectivity and is also antagonized by Nef.

Given SERINC5s role in viral infectivity, we hypothesized that the ability of non-Subtype B Nefs from PLWH in Uganda to downregulate SERINC5 would not correlate with latent reservoir decay rate, as de novo infections are not believed to occur in ART-suppressed individuals. We evaluated 49 primary Nef isolates using an infection-based assay and quantified SERINC5 downregulation by flow cytometry, revealing functional variability between isolates and individuals. As expected, this activity did not correlate with the rate of decay of the latent reservoir. However, we identified a significant inverse correlation between Nef-mediated MHC-I and SERINC5 downregulation (p=0.001). These findings will be further explored in an infectious viral yield assay and targeted mutagenesis.

Overall, we aim to characterize non-subtype B Nef’s ability to downregulate SERINC5 and its implications on the latent reservoir in those living with HIV-1.

Background: The intestinal environment sustains a state of HIV latency via yet unclear mechanisms. We demonstrated that Th17 cytokines differentially act on intestinal epithelial cells (IEC) to promote viral reservoir (VR) latency (IL-22) or reactivation (IL-17A) in CD4+ T-cells of people with HIV (PWH) on antiretroviral therapy (ART). The VR modulation by IL-22/IL-17A coincided with the up/down-regulation of IL-32, a cytokine with pro-inflammatory/antiviral features overexpressed in ART-treated PWH. Since IL-32 is an interferon (IFN) stimulated gene, we investigated IFN-β1a ability to modulate the IEC:T crosstalk via IL-32-dependent mechanisms.
Methods: CRISPR-Cas9 was used to inactivate IL32 in HT-29 IEC. IL-32 mRNA/protein expression was quantified by real-time RT-PCR/ELISA. CD4+ T-cells were isolated by magnetic beads negative selection. IEC were exposed to IFN-β1a and/or TNF prior being co-cultured with CD3/CD28-activated CD4+ T-cells of male ART-treated PWH (n=6) for 12 days. Intracellular/soluble HIV-p24 were quantified by flow cytometry/ELISA. Integrated HIV-DNA was quantified by PCR. Friedman and Dunn’s post-test were applied. The Illumina RNA-Sequencing identified differentially-expressed genes (DEG) based on adjusted p-values and fold changes, and pathways based on Gene Set Variation Analysis (GSEA).
Results: Control but not IL32 knockout IEC upregulated IL-32 mRNA/protein upon IFN+TNF exposure. Regardless of IL-32 expression, HIV outgrowth was significantly reduced when CD4+ T-cells of ART-treated PWH where co-cultured with IFN+TNF-stimulated IEC. The decreased HIV outgrowth was associated with a slight reduced HIV-DNA integration levels, without changes in T-cell viability/proliferation. Transcriptional profiling in IFN+TNF-exposed IEC identified potential mediators shaping the IEC:T crosstalk, including IFN lambda 2 (IFNL2), CD274/PD-L1, CD83, CD223/LAG3, and LGALS9/Galectin9.
Conclusions: We demonstrated that IFN-β1a transcriptionally reprograms TNF-activated IEC for sustaining HIV latency in CD4+ T-cells of ART-treated PWH via mechanisms independent of IL-32, but likely involving IFNL2, PD-L1, and other immune checkpoints. Thus, the IEC:T crosstalk may be pharmacologically and/or immunologically targeted in novel HIV cure interventions.

Introduction
The major obstacle to HIV eradication is the persistence of viral genomes in long-lived CD4+ T cells. Emerging data indicate that most proviruses remain transcriptionally active in people with HIV (PWH) on ART. However, the nature of the HIV transcripts produced and whether they can encode viral proteins remain unclear. We developed a novel approach to analyse the viral transcriptome of untouched HIV reservoir cells and following stimulation with several well-characterized latency-reversing agents (LRAs).
Methods
Cellular RNAs were extracted from CD4+ T cells obtained from four ART-treated PWH. Cells were either unstimulated or stimulated for 18 hours with PMA/ionomycin, romidepsin, interleukin-15 or anti-CD3/CD28. In vitro HIV-infected cells were used as positive controls. Viral RNAs were reverse-transcribed, labeled with unique molecular identifiers, amplified, and sequenced using the Oxford Nanopore Technology pipeline.
Results
Consensus RNA profiles derived from in vitro-infected cells were consistent with previously published data, thereby validating our experimental approach. Unstimulated CD4+ T cells from PWH on ART contained exclusively non-coding RNAs, consistent with residual transcription from defective proviruses. In contrast, stimulation with LRAs induced the production of coding transcripts in variable proportions (20% to 93% of all viral RNAs, with increasing proportions from romidepsin, IL-15, CD3/CD28 and PMA/ionomycin). Most canonical donor and acceptor splicing sites were used by coding transcripts, with the exception of D1. Instead, 12 alternate D1 sites were newly identified. Among those, a recurrent alternative D1 site located upstream of the canonical splice donor site (position 583) was used by a mean of 65% of all coding viral transcripts across all compounds and participants.
Conclusion
Our results indicate that most HIV transcripts spontaneously produced in PWH on ART cannot be translated into functional proteins. Upon stimulation, we uncovered a previously underestimated diversity of atypical viral RNA splicing, revealing the heterogeneity of the viral reservoir.