Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and mucosal reservoirs

Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure

Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir

HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01 ± 0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA

In-depth validation of total HIV-1 DNA assays for quantification of various HIV-1 subtypes

HIV-1 DNA quantification serves as an important reservoir biomarker in HIV cure trials. However, the high genetic diversity of HIV-1 represented by different subtypes may bring inaccuracy in quantifying HIV-1 DNA and a sensitive and validated assay covering diverse HIV-1 subtypes is lacking. Therefore, we cross-validated total HIV-1 DNA assays described in literature using a three-step comparative analysis. First, a bioinformatics tool was developed in-house to perform an in silico evaluation of 67 HIV-1 DNA assays. Secondly, these selected assays were in vitro validated using a panel of different HIV-1 subtypes and, finally, ex vivo assessed on selected patient samples with different HIV-1 subtypes. Our results show that quantification of HIV-1 DNA substantially differs between assays and we advise five best performing HIV-1 DNA assays for ddPCR and qPCR (Schvachsa_2007, Viard_2004, Heeregrave_2009, Van_der_Sluis_2013, Yu_2008 and Yun_2002). This in-depth analysis of published HIV-1 DNA assays indicates that not all assays guarantee an optimal measurement of HIV-1 DNA, especially when looking across subtypes. Using an in-depth cross-validation, we were able to validate HIV-1 DNA assays that are suitable for quantification of HIV-1 DNA in a wide variety of HIV-1 infected patients

Early treated HIV-1 positive individuals demonstrate similar restriction factor expression profile as long-term non-progressors

Background: A wide range of host restriction factors (RF) become upregulated upon HIV-1 infection to suppress viral infectivity and may aid viremic control in vivo. This cross-sectional study evaluated HIV-1 RFs and dependency factors in HIV infected individuals with progressive or non-progressive infection, as well as in early and late treated cohorts that exhibit different viro-immunological profiles due to differences in timing of treatment-initiation. Methods: The expression profile of IFIT1, MX1, APOBEC3G, SAMHD1, BST2 (encoding TETHERIN), TRIM5, MX2, SLFN11, PAF1, PSIP1 (encoding LEDGF/p75), and NLRX1 was measured by qPCR in 104 HIV-1 positive individuals: seroconverters (SRCV; n =19), long term non-progressors (LTNP; n =17), viremic progressors (VP; n =12), patients treated during seroconversion (Early treated; n =24) or chronic infection (Late treated; n =32), and non-infected controls. Findings: Expression levels of early treated HIV-1 positive individuals were significantly upregulated in comparison to late treated patients (IFIT1: p=0.0003; MX1: p=0.008; APOBEC3G: p=0.002; SAMHD1: p=0.0008; SLFN11: p<0.0001; BST2: p<0.0001). Similarly, SLFN11, BST2, and SAMHD1 were highly expressed in LTNPs at comparable levels as in early treated HIV-1 positive individuals. Furthermore, SLFN11 and SAMHD1 expression negatively correlated with total and integrated HIV-1 DNA levels. Interpretation: Early treatment initiation maintains initial RF elevation even after a decade of ART. Elevated expression of SLFN11, BST2, and SAMHD1 in LTNP and early treated subjects implies that these RFs may be associated with spontaneous virological control

Comparison of methods for in-house screening of HLA*B57:01 to prevent abacavir hypersensitivity in HIV-1 care

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting

Comprehensive characterization of LEDGF/p75 in a HIV-1-infected patient cohort

BACKGROUND: Lens epithelium derived growth factor/transcriptional co-activator p75 (LEDGF/p75) is an important cellular co-factor for the HIV enzyme integrase. In the present study, we evaluated if genetic variation in the LEDGF/p75 gene and mRNA expression levels might explain differences in HIV disease progression. METHODS: Samples were derived from a therapy-naïve patient cohort from the Ghent University Hospital and from the long-term-non-progressor patient Spanish RIS cohort. A comprehensive genomic scan of the coding region and 3’UTR of LEDGF/p75 was performed using high resolution melting curve analysis and Sanger sequencing to identify single nucleotide polymorphisms (SNPS). In addition, LEDGF/p75 mRNA expression levels were determined from patient PBMCs using RT-qPCR with validated reference genes for normalization. RESULTS: In total 325 patient samples were investigated, of which 291 (90%) of Caucasian and 34 (10%) of African origin, and among which a large group of Elite controllers (n=49) and Viremic controllers (n=62). In these samples, 24 SNPs were analyzed, including 5 in the coding region (2 synonymous and 3 non-synonymous), 17 in the flanking non-coding regions and in the 3’UTR, and two additional tagSNPs as described by Madlala et al. (Aids, 2011) in two South African cohorts. One SNP in the 3’UTR region (rs2737835, n=46) had a higher representation in Caucasian Elite controllers and was correlated with lower LEDGF/p75 mRNA levels (P=0.047) and with a slower CD4 decline (P= 0.042). rs2737828 (n=13) was under-represented in Caucasian HIV patients and linked to lower LEDGF/p75 expression (P=0.013). The presence of intron SNP (rs16933270, n=6) was associated with a slower CD4 decline in African patients (P=0.017), and this CD4 decline was comparable with that of African slow disease progressors. Interestingly, the presence of one tagSNP (rs12339417, n=95) was significantly correlated with a decreased viral load, but in contrast to the results of Madlala et al. (Aids, 2011), this SNP was not correlated with the CD4 slope and neither with LEDGF/p75 mRNA levels. CONCLUSIONS: Although the data of the present investigation was not entirely comparable with the results of Madlala et al. (Aids, 2011), our data supports their hypothesis that host factors influence HIV disease progression. The observed differences between the European and South African cohorts may be of ethnical origin, or due to different infection phases. In the investigated cohorts, two SNPs were associated with lower LEDGF/p75 mRNA expression in Caucasians, and one SNP was associated with slower disease progression in Africans. The significant correlation with the tagSNP (rs12339417) and the decreased viral load is surprising as this was not correlated with a delayed CD4 decline, nor with LEDGF/p75 expression. This might indicate that either conformational changes or factors upstream of mRNA transcription might influence the action of LEDGF/p75 in these HIV patients

Characterization of LEDGF/p75 genetic variants and association with HIV-1 disease progression

BACKGROUND: As Lens epithelium-derived growth factor (LEDGF/p75) is an important co-factor involved in HIV-1 integration, the LEDGF/p75-IN interaction is a promising target for the new class of allosteric HIV integrase inhibitors (LEDGINs). Few data are available on the genetic variability of LEDGF/p75 and the influence on HIV disease in vivo. This study evaluated the relation between LEDGF/p75 genetic variation, mRNA expression and HIV-1 disease progression in order to guide future clinical use of LEDGINs. METHODS: Samples were derived from a therapy-naïve cohort at Ghent University Hospital and a Spanish long-term-non-progressor cohort. High-resolution melting curve analysis and Sanger sequencing were used to identify all single nucleotide polymorphisms (SNPs) in the coding region, flanking intronic regions and full 3'UTR of LEDGF/p75. In addition, two intronic tagSNPs were screened based on previous indication of influencing HIV disease. LEDGF/p75 mRNA was quantified in patient peripheral blood mononuclear cells (PBMC) using RT-qPCR. RESULTS: 325 samples were investigated from patients of Caucasian (n = 291) and African (n = 34) origin, including Elite (n = 49) and Viremic controllers (n = 62). 21 SNPs were identified, comprising five in the coding region and 16 in the non-coding regions and 3'UTR. The variants in the coding region were infrequent and had no major impact on protein structure according to SIFT and PolyPhen score. One intronic SNP (rs2737828) was significantly under-represented in Caucasian patients (P<0.0001) compared to healthy controls (HapMap). Two SNPs showed a non-significant trend towards association with slower disease progression but not with LEDGF/p75 expression. The observed variation in LEDGF/p75 expression was not correlated with disease progression. CONCLUSIONS: LEDGF/p75 is a highly conserved protein. Two non-coding polymorphisms were identified indicating a correlation with disease outcome, but further research is needed to clarify phenotypic impact. The conserved coding region and the observed variation in LEDGF/p75 expression are important characteristics for clinical use of LEDGINs.This work was partly supported by the Flemish Agency for Innovation by Science and Technology (CellCoVir - IWT file nr 60813). Linos Vandekerckhove is supported by the National Fund for Scientific Research – Belgium as Principal Investigator. The Spanish RIS cohort and HIV BioBank are integrated in the Spanish AIDS Research Network supported by Instituto de Salud Carlos III (Grant RD06/0006/0035) and Fundación para la Investigación y Prevención del SIDA en España (FIPSE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S