Active PD-L1 incorporation within HIV virions functionally impairs T follicular helper cells.

The limited development of broadly neutralizing antibodies (BnAbs) during HIV infection is classically attributed to an inadequate B-cell help brought by functionally impaired T follicular helper (Tfh) cells. However, the determinants of Tfh-cell functional impairment and the signals contributing to this condition remain elusive. In the present study, we showed that PD-L1 is incorporated within HIV virions through an active mechanism involving p17 HIV matrix protein. We subsequently showed that in vitro produced PD-L1high but not PD-L1low HIV virions, significantly reduced Tfh-cell proliferation and IL-21 production, ultimately leading to a decreased of IgG1 secretion from GC B cells. Interestingly, Tfh-cell functions were fully restored in presence of anti-PD-L1/2 blocking mAbs treatment, demonstrating that the incorporated PD-L1 proteins were functionally active. Taken together, the present study unveils an immunovirological mechanism by which HIV specifically exploits the regulatory potential of PD-L1 to suppress the immune system during the course of HIV infection.

Lymph node migratory dendritic cells modulate HIV-1 transcription through PD-1 engagement.

T-follicular helper (Tfh) cells, co-expressing PD-1 and TIGIT, serve as a major cell reservoir for HIV-1 and are responsible for active and persistent HIV-1 transcription after prolonged antiretroviral therapy (ART). However, the precise mechanisms regulating HIV-1 transcription in lymph nodes (LNs) remain unclear. In the present study, we investigated the potential role of immune checkpoint (IC)/IC-Ligand (IC-L) interactions on HIV-1 transcription in LN-microenvironment. We show that PD-L1 (PD-1-ligand) and CD155 (TIGIT-ligand) are predominantly co-expressed on LN migratory (CD1chighCCR7+CD127+) dendritic cells (DCs), that locate predominantly in extra-follicular areas in ART treated individuals. We demonstrate that TCR-mediated HIV production is suppressed in vitro in the presence of recombinant PD-L1 or CD155 and, more importantly, when LN migratory DCs are co-cultured with PD-1+/Tfh cells. These results indicate that LN migratory DCs expressing IC-Ls may more efficiently restrict HIV-1 transcription in the extra-follicular areas and explain the persistence of HIV transcription in PD-1+/Tfh cells after prolonged ART within germinal centers.

Detection of antisense protein (ASP) RNA transcripts in individuals infected with human immunodeficiency virus type 1 (HIV-1).

The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not allow assessment of the polarity of the initial RNA template, causing the amplification of non-specific cDNAs. In this study we have developed a modified protocol for the detection of human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) RNA. Using this approach, we have identified ASP transcripts in CD4+ T cells isolated from five HIV-infected individuals, either untreated or under suppressive therapy. We show that ASP RNA can be detected in stimulated CD4+ T cells from both groups of patients, but not in unstimulated cells. We also show that in untreated patients, the patterns of expression of ASP and env are very similar, with the levels of ASP RNA being markedly lower than those of env. Treatment of cells from one viraemic patient with α-amanitin greatly reduces the rate of ASP RNA synthesis, suggesting that it is associated with RNA polymerase II, the central enzyme in the transcription of protein-coding genes. Our data represent the first nucleotide sequences obtained in patients for ASP, demonstrating that its transcription indeed occurs in those HIV-1 lineages in which the ASP open reading frame is present.

CD32+ and PD-1+ Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals.

A recent study conducted in blood has proposed CD32 as the marker identifying the "elusive" HIV reservoir. We have investigated the distribution of CD32+ CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1+ CD4 T cells. The frequency of CD32+ CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32+ cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32+ CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32+ and PD-1+ CD4 T cells compared to CD32- and PD-1- cells in both viremic and treated individuals, but there was no difference between CD32+ and PD-1+ cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32+ versus PD-1+ cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32+ PD-1+ CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32- PD-1- (averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32+ PD-1- (2.2-fold in treated individuals and 4.3-fold in viremics), and CD32- PD-1+ (2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32+ PD-1- and CD32- PD-1+ CD4 T cells. Interestingly, the proportion of CD32+ and PD-1+ CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

In Vitro Reactivation of Replication-Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors.

UNLABELLED: The existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latency in vitro. Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects. IMPORTANCE: The major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an experimental strategy that would allow the evaluation of latency-reversing agent (LRA) efficiency in vitro using primary CD4 T cells. In the present study, we demonstrate that HDACis are potent inducers of replication-competent and infectious HIV-1 in resting memory CD4 T cells of long-term ART-treated patients and identify givinostat as the most efficient LRA tested.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy.

UNLABELLED: Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT01365065.

Myeloid dendritic cells induce HIV-1 latency in non-proliferating CD4+ T cells.

Latently infected resting CD4(+) T cells are a major barrier to HIV cure. Understanding how latency is established, maintained and reversed is critical to identifying novel strategies to eliminate latently infected cells. We demonstrate here that co-culture of resting CD4(+) T cells and syngeneic myeloid dendritic cells (mDC) can dramatically increase the frequency of HIV DNA integration and latent HIV infection in non-proliferating memory, but not naïve, CD4(+) T cells. Latency was eliminated when cell-to-cell contact was prevented in the mDC-T cell co-cultures and reduced when clustering was minimised in the mDC-T cell co-cultures. Supernatants from infected mDC-T cell co-cultures did not facilitate the establishment of latency, consistent with cell-cell contact and not a soluble factor being critical for mediating latent infection of resting CD4(+) T cells. Gene expression in non-proliferating CD4(+) T cells, enriched for latent infection, showed significant changes in the expression of genes involved in cellular activation and interferon regulated pathways, including the down-regulation of genes controlling both NF-κB and cell cycle. We conclude that mDC play a key role in the establishment of HIV latency in resting memory CD4(+) T cells, which is predominantly mediated through signalling during DC-T cell contact.

Cutting edge: Prolonged exposure to HIV reinforces a poised epigenetic program for PD-1 expression in virus-specific CD8 T cells.

Ag-specific CD8 T cells play a critical role in controlling HIV infection but eventually lose antiviral functions in part because of expression and signaling through the inhibitory programmed death-1 (PD-1) receptor. To better understand the impact of prolonged TCR ligation on regulation of PD-1 expression in HIV-specific CD8 T cells, we investigated the capacity of virus-specific CD8 T cells to modify the PD-1 epigenetic program after reduction in viral load. We observed that the transcriptional regulatory region was unmethylated in the PD-1(hi) HIV-specific CD8 T cells, whereas it remained methylated in donor-matched naive cells at acute and chronic stages of infection. Surprisingly, the PD-1 promoter remained unmethylated in HIV-specific CD8 T cells from subjects with a viral load controlled by antiviral therapy for >2 y or from elite controllers. Together, these data demonstrate that the epigenetic program at the PD-1 locus becomes fixed after prolonged exposure to HIV virus.

Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection.

Immediate-early host-virus interactions that occur during the first weeks after HIV infection have a major impact on disease progression. The mechanisms underlying the failure of HIV-specific CD8 T-cell response to persist and control viral replication early in infection are yet to be characterized. In this study, we performed a thorough phenotypic, gene expression and functional analysis to compare HIV-specific CD8 T cells in acutely and chronically infected subjects. We showed that HIV-specific CD8 T cells in primary infection can be distinguished by their metabolic state, rate of proliferation, and susceptibility to apoptosis. HIV-specific CD8 T cells in acute/early HIV infection secreted less IFN-γ but were more cytotoxic than their counterparts in chronic infection. Importantly, we showed that the levels of IL-7R expression and the capacity of HIV-specific CD8 T cells to secrete IL-2 on antigenic restimulation during primary infection were inversely correlated with the viral set-point. Altogether, these data suggest an altered metabolic state of HIV-specific CD8 T cells in primary infection resulting from hyperproliferation and stress induced signals, demonstrate the discordant function of HIV-specific CD8 T cells during early/acute infection, and highlight the importance of T-cell maintenance for viral control.

A scalable workflow to test "shock and kill" therapeutic approaches against the HIV-1 latent reservoir in blood cells ex vivo.

Integrated HIV-1 DNA persists in cells of people living with HIV during antiretroviral treatment, but its quantification is hindered by its rarity. Here, we present an optimized protocol to evaluate "shock and kill" therapeutic strategies, including both the latency reactivation ("shock") and elimination of infected cells ("kill") stages. We describe steps for the sequential use of nested PCR-based assays and viability sorting to allow for scalable and rapid screening of candidate therapeutics in patient-derived blood cells. For complete details on the use and execution of this protocol, please refer to Shytaj et al..1.

Lymph node dendritic cells harbor inducible replication-competent HIV despite years of suppressive ART.

Although gut and lymph node (LN) memory CD4 T cells represent major HIV and simian immunodeficiency virus (SIV) tissue reservoirs, the study of the role of dendritic cells (DCs) in HIV persistence has long been limited to the blood due to difficulties to access lymphoid tissue samples. In this study, we show that LN migratory and resident DC subpopulations harbor distinct phenotypic and transcriptomic profiles. Interestingly, both LN DC subpopulations contain HIV intact provirus and inducible replication-competent HIV despite the expression of the antiviral restriction factor SAMHD1. Notably, LN DC subpopulations isolated from HIV-infected individuals treated for up to 14 years are transcriptionally silent but harbor replication-competent virus that can be induced upon TLR7/8 stimulation. Taken together, these results uncover a potential important contribution of LN DCs to HIV infection in the presence of ART.

Differential Production of Midkine and Pleiotrophin by Innate APCs upon Stimulation through Nucleic Acid-Sensing TLRs.

Midkine (MK) and pleiotrophin (PTN) belong to the same family of cytokines. They have similar sequences and functions. Both have important roles in cellular proliferation, tumors, and diseases. They regulate and are expressed by some immune cells. We have recently demonstrated MK production by some human innate antigen-presenting cells (iAPCs), i.e., monocyte-derived dendritic cells (MDDCs) and macrophages stimulated through Toll-like receptor (TLR)-4, and plasmacytoid dendritic cells (pDCs) stimulated through TLR 7. While PTN production was only documented in tissue macrophages. TLRs 3, 7, 8, and 9 are nucleic acid sensing (NAS) TLRs that detect nucleic acids from cell damage and infection and induce iAPC responses. We investigated whether NAS TLRs can induce MK and PTN production by human iAPCs, namely monocytes, macrophages, MDDCs, myeloid dendritic cells (mDCs), and pDCs. Our results demonstrated for the first time that PTN is produced by all iAPCs upon TLR triggering (p < 0.01). IAPCs produced more PTN than MK (p < 0.01). NAS TLRs and iAPCs had differential abilities to induce the production of MK, which was induced in monocytes and pDCs by all NAS TLRs (p < 0.05) and in MDDCs by TLRs 7/8 (p < 0.05). TLR4 induced a stronger MK production than NAS TLRs (p ≤ 0.05). Monocytes produced higher levels of PTN after differentiation to macrophages and MDDCs (p < 0.05). The production of MK and PTN differs among iAPCs, with a higher production of PTN and a selective induction of MK production by NAS TLR. This highlights the potentially important role of iAPCs in angiogenesis, tumors, infections, and autoimmunity through the differential production of MK and PTN upon TLR triggering.

TILDA: Tat/Rev Induced Limiting Dilution Assay.

Recently the Tat/rev Induced Limiting Dilution Assay, or TILDA, has been proposed as a possible alternative method to quantify the HIV-1 reservoir. TILDA estimates the frequency of latently infected cells by probing, in a limiting dilution format, the presence or inducibility of tat and rev multiply spliced HIV-1 RNA. In doing so, TILDA reduces overestimation of reservoir size compared to HIV-1 DNA measurements because multiply spliced HIV-1 RNA is less likely to be transcribed from dysfunctional genomes with replication defects. TILDA is easy to perform, requires a very low input number of cells and has a fast turnaround time, making it ideal for use in clinical settings. Here we describe the execution of TILDA with particular emphasis on cell preparation and the limiting dilution scheme.

The deficiency in Th2-like Tfh cells affects the maturation and quality of HIV-specific B cell response in viremic infection.

Optimal T follicular helper (Tfh) cells function is important to promote the development of germinal centers and maturation of high affinity antigen-specific B cells. We have found that the expression of CXCR3 defines distinct Tfh subsets: CXCR3+ Th1-like Tfh cells mainly producing single IFN-γ and dual IL-21/IFN-γ and CXCR3- Th2-like Tfh cells mainly producing single IL-4 and dual IL-21/IL-4 cytokines. CXCR3- Th2-like Tfhs are significantly reduced during ongoing HIV replication. While the percentage of Th2-like Tfh cells correlates with that of total and cycling HIV-specific B cells, the percentage of CXCR3+ Th1-like Tfhs correlates with HIV-specific B cells expressing T-bet and CXCR3. Of note, only IL-4 and IL-21 cytokines boosted efficient maturation of HIV-specific B cells while IFN-γ induced expression of T-bet and CXCR3 in B cells. Interestingly, total and HIV-specific CXCR3+ B cells showed lower rate of somatic hypermutation, as compared to CXCR3- B cells. Therefore, the imbalance in Th2/Th1-like Tfhs affects B cell responses in viremic HIV infection.

Human macrophages and monocyte-derived dendritic cells stimulate the proliferation of endothelial cells through midkine production.

The cytokine midkine (MK) is a growth factor that is involved in different physiological processes including tissue repair, inflammation, the development of different types of cancer and the proliferation of endothelial cells. The production of MK by primary human macrophages and monocyte-derived dendritic cells (MDDCs) was never described. We investigated whether MK is produced by primary human monocytes, macrophages and MDDCs and the capacity of macrophages and MDDCs to modulate the proliferation of endothelial cells through MK production. The TLR stimulation of human monocytes, macrophages and MDDCs induced an average of ≈200-fold increase in MK mRNA and the production of an average of 78.2, 62, 179 pg/ml MK by monocytes, macrophages and MDDCs respectively (p < 0.05). MK production was supported by its detection in CD11c+ cells, CLEC4C+ cells and CD68+ cells in biopsies of human tonsils showing reactive lymphoid follicular hyperplasia. JSH-23, which selectively inhibits NF-κB activity, decreased the TLR-induced production of MK in PMBCs, macrophages and MDDCs compared to the control (p < 0.05). The inhibition of MK production by macrophages and MDDCs using anti-MK siRNA decreased the capacity of their supernatants to stimulate the proliferation of endothelial cells (p = 0.01 and 0.04 respectively). This is the first study demonstrating that the cytokine MK is produced by primary human macrophages and MDDCs upon TLR triggering, and that these cells can stimulate endothelial cell proliferation through MK production. Our results also suggest that NF-κB plays a potential role in the production of MK in macrophages and MDDCs upon TLR stimulation. The production of MK by macrophages and MDDCs and the fact that these cells can enhance the proliferation of endothelial cells by producing MK are novel immunological phenomena that have potentially important therapeutic implications.

Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA.

The presence of a stable HIV-1 reservoir persisting over time despite effective antiretroviral suppression therapy precludes a cure for HIV-1. Characterizing and quantifying this residual reservoir is considered an essential prerequisite to develop and validate curative strategies. However, a sensitive, reproducible, cost-effective, and easily executable test is still needed. The quantitative viral outgrowth assay is considered the gold standard approach to quantify the reservoir in HIV-1-infected patients on suppressive ART, but it has several limitations. An alternative method to quantify the viral reservoir following the reactivation of latent HIV-1 provirus detects multiply-spliced tat/rev RNA (msRNA) molecules by real-time PCR [tat/rev induced limiting dilution assay (TILDA)]. This article provides a perspective overview of the clinical relevance, various applications, recent advancements of TILDA, and how the assay has contributed to our understanding of the HIV-1 reservoir.

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress.

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic, and metabolomic analyses, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+ /NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a "shock and kill effect" decreasing proviral DNA in cells from people living with HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.

Inter-Laboratory Reproducibility of Inducible HIV-1 Reservoir Quantification by TILDA.

Substantial efforts to eliminate or reduce latent HIV-1 reservoirs are underway in clinical trials and have created a critical demand for sensitive, accurate, and reproducible tools to evaluate the efficacy of these strategies. Alternative reservoir quantification assays have been developed to circumvent limitations of the quantitative viral outgrowth assay. One such assay is tat/rev induced limiting dilution assay (TILDA), which measures the frequency of CD4+ T cells harboring inducible latent HIV-1 provirus. We modified pre-amplification reagents and conditions (TILDA v2.0) to improve assay execution and first internally validated assay performance using CD4+ T cells obtained from cART-suppressed HIV-1-infected individuals. Detection of tat/rev multiply spliced RNA was not altered by modifying pre-amplification conditions, confirming the robustness of the assay, and supporting the technique's amenability to limited modifications to ensure better implementation for routine use in clinical studies of latent HIV-1 reservoirs. Furthermore, we cross-validated results of TILDA v2.0 and the original assay performed in two separate laboratories using samples from 15 HIV-1-infected individuals. TILDA and TILDA v2.0 showed a strong correlation (Lin's Concordance Correlation Coefficient = 0.86). The low inter-laboratory variability between TILDAs performed at different institutes further supports use of TILDA for reservoir quantitation in multi-center interventional HIV-1 Cure trials.

Comparative analysis and generation of a robust HIV-1 DNA quantification assay.

HIV-1 infection cannot be cured due to the presence of the latent reservoir (LR). Novel cure or treatment strategies, such as "shock and kill" or therapeutic vaccination, aim to reduce or eradicate the LR. Cure strategies utilise robust DNA quantification assays to measure the change in the LR in low copy scenarios. No standard assay exists, which impedes the reliable comparison of results from different therapy and vaccine trials and HIV-1 total DNA quantification methods have not been previously compared. The HIV-1 long terminal repeat (LTR) has been shown to be the best target for DNA quantification. We have analysed two HIV-1 quantification assays, both able to differentiate between the variant HIV-1 DNA forms via the use of pre-amplification and primers targeting LTR. We identify a strong correlation (r=0.9759, P<0.0001) between assays which is conserved in low copy samples (r=0.8220, P<0.0001) indicating that these assays may be used interchangeably. The RvS assay performed significantly (P=0.0021) better than the CV assay when quantifying HIV-1 total DNA in patient CD4+ T lymphocytes. Sequence analysis demonstrated that viral diversity can limit DNA quantification, however in silico analysis of the primers indicated that within the target region nucleotide miss-matches appear infrequently. Further in silico analysis using up to-date sequence information led to the improvement of primers and enabled us to establish a more broadly specific assay with significantly higher HIV-1 DNA quantification capacity in patient samples (p=0.0057, n=17).