Design, cohort profile and comparison of the KTD-Innov study: a prospective multidimensional biomarker validation study in kidney allograft rejection.

There is an unmet need for robust and clinically validated biomarkers of kidney allograft rejection. Here we present the KTD-Innov study (ClinicalTrials.gov, NCT03582436), an unselected deeply phenotyped cohort of kidney transplant recipients with a holistic approach to validate the clinical utility of precision diagnostic biomarkers. In 2018-2019, we prospectively enrolled consecutive adult patients who received a kidney allograft at seven French centers and followed them for a year. We performed multimodal phenotyping at follow-up visits, by collecting clinical, biological, immunological, and histological parameters, and analyzing a panel of 147 blood, urinary and kidney tissue biomarkers. The primary outcome was allograft rejection, assessed at each visit according to the international Banff 2019 classification. We evaluated the representativeness of participants by comparing them with patients from French, European, and American transplant programs transplanted during the same period. A total of 733 kidney transplant recipients (64.1% male and 35.9% female) were included during the study. The median follow-up after transplantation was 12.3 months (interquartile range, 11.9-13.1 months). The cumulative incidence of rejection was 9.7% at one year post-transplant. We developed a distributed and secured data repository in compliance with the general data protection regulation. We established a multimodal biomarker biobank of 16,736 samples, including 9331 blood, 4425 urinary and 2980 kidney tissue samples, managed and secured in a collaborative network involving 7 clinical centers, 4 analytical platforms and 2 industrial partners. Patients' characteristics, immune profiles and treatments closely resembled those of 41,238 French, European and American kidney transplant recipients. The KTD-Innov study is a unique holistic and multidimensional biomarker validation cohort of kidney transplant recipients representative of the real-world transplant population. Future findings from this cohort are likely to be robust and generalizable.

Validation of iQ-Check E. coli O157:H7 real-time PCR test kit for detection of Escherichia coli O157:H7 in selected foods.

iQ-Check E. coli O157:H7 (Bio-Rad Laboratories, Hercules, CA) is a real-time PCR kit for detection of E. coli O157:H7 from selected foods. Specific fluorescent oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These fluorescent probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. Three foods (ground beef, apple cider, fresh spinach) were selected to compare the performance of iQ-Check E. coli O157:H7 to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) reference method for ground beef and the U.S. Food and Drug Administration/Bacteriological Analytical Manual reference method for apple cider and fresh spinach. Three protocols were tested in this study: a shortened 8 h primary enrichment in buffered peptone water (BPW), a 24 h enrichment in BPW, and an enrichment in appropriate reference method enrichment broth. The iQ-Check E. coli O157:H7 method was able to identify more true/confirmed positive samples than the reference method. Inclusivity and exclusivity rates of the method were 100%. iQ-Check E. coli O157:H7 performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no significant difference observed in performance over the shelf life of the kit.

Is oxygen a key factor in the lipodystrophy phenotype?

BACKGROUND: The lipodystrophic syndrome (LD) is a disorder resulting from selective damage of adipose tissue by antiretroviral drugs included in therapy controlling human-immunodeficiency-virus-1. In the therapy cocktail the nucleoside reverse transcriptase inhibitors (NRTI) contribute to the development of this syndrome. Cellular target of NRTI was identified as the mitochondrial polymerase-gamma and their toxicity described as a mitochondrial DNA (mtDNA) depletion resulting in a mitochondrial cytopathy and involved in fat redistribution. No mechanisms offer explanation whatsoever for the lipo-atrophic and lipo-hypertrophic phenotype of LD. To understand the occurrence we proposed that the pO2 (oxygen partial pressure) could be a key factor in the development of the LD. For the first time, we report here differential effects of NRTIs on human adipose cells depending on pO2 conditions. RESULTS AND DISCUSSION: We showed that the hypoxia conditions could alter adipogenesis process by modifying expression of adipocyte makers as leptin and the peroxisome proliferator-activated receptor PPARgamma and inhibiting triglyceride (TG) accumulation in adipocytes. Toxicity of NRTI followed on adipose cells in culture under normoxia versus hypoxia conditions showed, differential effects of drugs on mtDNA of these cells depending on pO2 conditions. Moreover, NRTI-treated adipocytes were refractory to the inhibition of adipogenesis under hypoxia. Finally, our hypothesis that variations of pO2 could exist between adipose tissue from anatomical origins was supported by staining of the hypoxic-induced angiopoietin ANGPTL4 depended on the location of fat. CONCLUSION: Toxicity of NRTIs have been shown to be opposite on human adipose cells depending on the oxygen availability. These data suggest that the LD phenotype may be a differential consequence of NRTI effects, depending on the metabolic status of the targeted adipose tissues and provide new insights into the opposite effects of antiretroviral treatment, as observed for the lipo-atrophic and lipo-hypertrophic phenotype characteristic of LD.

A novel function for spumaretrovirus integrase: an early requirement for integrase-mediated cleavage of 2 LTR circles.

Retroviral integration is central to viral persistence and pathogenesis, cancer as well as host genome evolution. However, it is unclear why integration appears essential for retrovirus production, especially given the abundance and transcriptional potential of non-integrated viral genomes. The involvement of retroviral endonuclease, also called integrase (IN), in replication steps apart from integration has been proposed, but is usually considered to be accessory. We observe here that integration of a retrovirus from the spumavirus family depends mainly on the quantity of viral DNA produced. Moreover, we found that IN directly participates to linear DNA production from 2-LTR circles by specifically cleaving the conserved palindromic sequence found at LTR-LTR junctions. These results challenge the prevailing view that integrase essential function is to catalyze retroviral DNA integration. Integrase activity upstream of this step, by controlling linear DNA production, is sufficient to explain the absolute requirement for this enzyme. The novel role of IN over 2-LTR circle junctions accounts for the pleiotropic effects observed in cells infected with IN mutants. It may explain why 1) 2-LTR circles accumulate in vivo in mutants carrying a defective IN while their linear and integrated DNA pools decrease; 2) why both LTRs are processed in a concerted manner. It also resolves the original puzzle concerning the integration of spumaretroviruses. More generally, it suggests to reassess 2-LTR circles as functional intermediates in the retrovirus cycle and to reconsider the idea that formation of the integrated provirus is an essential step of retrovirus production.

Oxygen consumption by cultured human cells is impaired by a nucleoside analogue cocktail that inhibits mitochondrial DNA synthesis.

We evaluated oxygen consumption rates in human cells cultured in the presence of a nucleoside analog reverse transcriptase inhibitor (NRTI) cocktail that inhibits mitochondrial DNA synthesis. We treated a proliferating human lymphocyte cell line and a primary culture of human adipose cells with antiretroviral drugs (AZT+ddC+d4T). The effects of these drugs on mitochondrial DNA (mtDNA) levels and oxygen consumption rates were evaluated using semi-quantitative real-time PCR and an on-line monitoring Clark electrode system. We found that the NRTI treatment lowered oxygen consumption rates and inhibited mitochondrial DNA replication in human cell cultures. Inhibition of oxygen consumption was linearly proportional to inhibition of mtDNA replication. These results show for the first time that mitochondrial respiration is impaired in NRTI sensitive cells. The linear relationship between NRTI inhibition of respiration and NRTI inhibition of mtDNA replication indicates that small decreases in mtDNA levels can lead to respiratory deficits in the tissues of patients treated with anti-HIV drugs. We propose a model that takes into account the small differences in metabolic dynamics between peripheral and axial/visceral fat tissues. This model explains how NRTI-related respiratory deficits may lead to the presentation of opposing lipodystrophic syndromes in same patient.

Alu-LTR real-time nested PCR assay for quantifying integrated HIV-1 DNA.

An improved Alu-long terminal repeat (LTR) polymerase chain reaction (PCR) assay is described for the quantification of integrated HIV-1 DNA in infected cells. The method includes generation of an infected cell line containing numerous randomly distributed HIV-1 integrated DNA for the construction of the DNA standard and a two-step real-time PCR assay in which the first-round PCR amplifies the DNA sequence between the HIV-1 LTR and the nearest chromosomal Alu element, and the nested PCR specifically amplifies PCR products from the first-round PCR. This assay allows us to quantify proviral DNA with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents) and exhibits a broad range of quantification spanning 5 log10 provirus copies. This Alu-LTR-based real-time nested PCR assay may be particularly useful to quantify integrated HIV-1 DNA in patients. It may also allow for the precise study of integration of HIV-1 DNA or HIV-1 based lentiviral vectors and may be a valuable tool to test future inhibitors of integration.

Quantification of HFV-integrated DNA in human cells by Alu-LTR real-time PCR.

Integration is described as a key step in viral replication of all retroviruses. A sensitive and quantitative measure of an integrated molecule is a good way to examine the importance of the integration step and to evaluate efficiency of retroviral vectors for gene transfer or anti-integrase drugs. Here, we report a sensitive and quantitative real-time polymerase chain reaction (PCR) technique to measure integrated viral DNA in human cells during a foamy virus (HFV) infection. This technique is based on two steps of PCR. The first round amplifies Alu-LTR (long terminal repeat) sequences resulting from viral integration. The second round of PCR is performed to quantify these events of integration. Quantification is monitored by the comparison of the amplification curve of the sample against a standard scale constituted of viral DNA from chronically infected cells. Sensitivity of this technique allows us to detect as few as 25 copies of HFV-integrated DNA in 50,000 cells.

Longitudinal monitoring of 2-long terminal repeat circles in peripheral blood mononuclear cells from patients with chronic HIV-1 infection.

OBJECTIVE: To evaluate the potential use of 2-long terminal repeats (LTR) HIV circular DNA quantification for the monitoring of ongoing virus replication in treated HIV-1-infected patients. DESIGN AND METHODS: In a longitudinal setting, where the natural course of HIV-1 infection was in most cases disrupted by continuous or discontinuous antiviral therapy, 2-LTR circles of HIV-1 DNA were quantified in serial peripheral blood mononuclear cell samples, selected in retrospect from 16 patients with chronic HIV-1 infection, using quantitative real-time PCR. We compared variations of 2-LTR circle level with concomitant variations in plasma viral RNA level and with the frequency of productively infected cells and chromosome associated proviral DNA copy numbers in patient's peripheral blood mononuclear cells. RESULTS: Antiviral treatment led to a sharp decrease in plasma viraemia and infectious cell frequency. In contrast, we found that levels of proviral DNA and 2-LTR circles were significantly lower under treatment only when groups of specimens that were homogeneous, with respect both to plasma viraemia and infectious cell frequency, were compared. Moreover, during the time of undetectable plasma viraemia, scarcely any decline in proviral DNA or 2-LTR circle levels was observed. CONCLUSIONS: The low impact of antiviral treatment on 2-LTR circle levels in vivo, when plasma viraemia and infectious cell frequency both dramatically decline lead us to conclude that 2-LTR circles should not be used for the monitoring of recent viral replication in treated patients.

[Evolution and virulence of primate lentiviruses]. / Evolution et virulence des lentivirus de primates.

While the AIDS epidemic caused by human immunodeficiency viruses (HIV) has resulted in the death of over 20 million people worldwide, simian immunodeficiency virus (SIV) infection, found in numerous African primate species, does not induce disease symptoms. The factors accounting for this difference between humans and natural host of SIV remain poorly understood. The entangled nature of the host/virus relationship could be the answer, rather than independent virus or host factors. Such a relationship is as a consequence of host/virus adaptation which has evolved over long periods in naturally infected primate species.

Gag-specific immune enhancement of lentiviral infection after vaccination with an adenoviral vector in an animal model of AIDS.

The evaluation of vaccine strategies in animal models is essential for the development of a vaccine against HIV. In efficacy trials conducted in non-human primate models of AIDS, vaccines based on adenoviruses compared favourably with other vaccine vectors. To determine whether this strategy could be transposed to another animal model, and by extension, to humans, we have evaluated the efficacy of adenoviral vectors in a natural model of AIDS, infection of the cat by the feline immunodeficiency virus (FIV). Recombinant canine adenoviruses expressing the envelope glycoproteins or the Gag protein of a primary strain of FIV were constructed. Three groups of six cats were immunised twice with vectors expressing FIV antigens or with a vector expressing an irrelevant antigen, green fluorescent protein, by intramuscular and subcutaneous routes. Humoral responses were elicited against the transgene product in 6/6, 3/6 and 0/6 cats after immunisation against green fluorescent protein, Gag or the envelope glycoproteins, respectively. Six weeks after the second administration, cats were challenged by the intraperitoneal route with the homologous strain, and viral burden in plasma was followed by quantitative RT-PCR. Immunisation with FIV antigens did not afford protection. Rather, viral RNA was detected at earlier time points in cats immunised against Gag than in cats immunised with a vector expressing an irrelevant antigen. Such immune-mediated enhancement did not appear to have a long-range impact on viral set point or inversion of the CD4(+)/CD8(+) ratio. Thus, in the feline AIDS model pre-existing immunity against a viral antigen exacerbated acute phase infection.

Efficient and specific internal cleavage of a retroviral palindromic DNA sequence by tetrameric HIV-1 integrase.

BACKGROUND: HIV-1 integrase (IN) catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain.

The protozoan parasite Toxoplasma gondii enters hosts through the intestinal mucosa and colonizes distant tissues such as the brain, where its progeny persists for a lifetime. We investigated the role of CD11c- and CD11b-expressing leukocytes in T. gondii transport during the early step of parasitism from the mouse small intestine and during subsequent parasite localization in the brain. Following intragastric inoculation of cyst-containing parasites in mice, CD11c+ dendritic cells from the intestinal lamina propria, the Peyer patches, and the mesenteric lymph nodes were parasitized while in the blood, parasites were associated with the CD11c- CD11b+ monocytes. Using adoptive transfer experiments, we demonstrated that these parasitized cells triggered a parasitic process in the brain of naive recipient mice. Ex vivo analysis of parasitized leukocytes showed that single tachyzoites remained at the cell periphery, often surrounded by the host cell plasma membrane, but did not divide. Using either a dye that labels circulating leukocytes or an antibody known to prevent CD11b+ circulating leukocytes from leaving the microvascular bed lumen, and chimeric mice in which the hematopoietic cells expressed the green fluorescent protein, we established that T. gondii zoites hijacked CD11b+ leukocytes to reach the brain extravascular space.

Inhibition of early steps of HIV-1 replication by SNF5/Ini1.

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.

Evaluation of live feline immunodeficiency virus vaccines with modified antigenic properties.

Live-attenuated viruses have typically been generated from pathogenic viruses by genetic modifications that modified their replicative capacity. The present study investigated whether modification of the antigenic properties of live-attenuated viruses might improve upon the protection that such vaccines afford against lentivirus infection. In a previous study, random amino acid substitutions were introduced into the transmembrane envelope glycoprotein of the feline immunodeficiency virus (FIV), within a highly conserved domain (principal immunodominant domain) bearing immunodominant B-cell epitopes. Amongst a wide set of mutants, mutations that modified antibody specificity without abolishing infectivity ex vivo were selected. In the present study, two such mutants, TN14 and TN92, were evaluated for their replicative capacities and pathogenic properties in vivo in comparison with the parental virus, FIV 34TF10. No significant differences in viral load were observed between mutant and parental viruses. After 1 year of infection, all animals were subjected to a heterologous intraclade superinfection with a primary strain of FIV. Whilst both parental and modified viruses protected cats from high viral loads after superinfection, the TN92 virus afforded a higher degree of protection (P=0.0079). Such improvement in protection might correlate with a decrease in the immunogenicity of a B-cell epitope potentially involved in antibody enhancement of infection.

Evidence for gene expression by unintegrated human immunodeficiency virus type 1 DNA species.

The integrated form of human immunodeficiency virus type 1 (HIV-1) DNA is classically considered to be the sole template for viral gene expression. However, several studies have suggested that unintegrated viral DNA species could also support transcription. To determine the contribution of the different species of HIV-1 DNA to viral expression, we first monitored intracellular levels of various HIV-1 DNA and RNA species in a single-round infection assay. We observed that, in comparison to the precocity of HIV-1 DNA synthesis, viral expression was delayed, suggesting that only the HIV-1 DNA species that persist for a sufficient period of time would be transcribed efficiently. We next evaluated the transcriptional activity of the circular forms of HIV-1 DNA bearing two long terminal repeats, since these episomes were reported to exhibit an intrinsic molecular stability. Our results support the notion that these circular species of HIV-1 DNA are naturally transcribed during HIV-1 infection, thereby participating in virus replication.

Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus.

A novel Alu-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication.

Biphasic DNA synthesis in spumaviruses.

Spumaviruses are complex retroviruses whose replication cycle resembles that of hepadnaviruses, especially by a late-occurring reverse transcription step. The possible existence of an early reverse transcription as observed in other retroviruses was not documented. Using real-time quantitative PCR, we addressed directly the kinetics of DNA synthesis during spumavirus infection. An early phase of viral DNA synthesis developed until 3 h postinfection, followed by a second phase, culminating 10 h postinfection. Both phases were abolished by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. Similar to other retroviruses, circular forms of viral DNA harboring two long terminal repeats were mainly found in the nucleus of infected cells. Interestingly, a fraction of these circular forms were detected in the cytoplasm and in extracellular virions, a feature shared with hepadnaviruses. Combined with packaging of both viral DNA and RNA genomes in virions, early and late reverse transcription might allow spumavirus to maximize its genome replication.

Cell cycle regulation of human immunodeficiency virus type 1 integration in T cells: antagonistic effects of nuclear envelope breakdown and chromatin condensation.

We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression.

Quantitation of blood lymphocyte mitochondrial DNA for the monitoring of antiretroviral drug-induced mitochondrial DNA depletion.

OBJECTIVE: To investigate the impact of antiretroviral treatment on the mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients. DESIGN: As absolute mtDNA copy numbers widely differ between individuals, we performed a longitudinal analysis where the patient's first historical specimen was obtained as a baseline reference for relative comparison with subsequent samples from that patient. METHODS: mtDNA and nuclear DNA quantitation per cell (beta-globin gene copies) were both measured by real-time polymerase chain reaction analysis of whole DNA extracts of 361 serial live-cryopreserved PBMCs collected in former trials and clinical follow-ups from 60 individuals with established or recently acquired HIV-1 infections before and during administration of various antiviral combination therapies. RESULTS: mtDNA amounts were stable or increasing over years of natural HIV-1 infection in untreated patients (n = 7), consistent with our finding of a lack of differences in mtDNA copy numbers in patients with either a long established or recent lentivirus infection. Our quantitation system revealed significant changes in mtDNA copy number depending on the designated triple, quadruple, or quintuple anti-HIV drug combinations. Zidovudine + zalcitabine + ritonavir and zidovudine + lamivudine + didanosine regularly lead to mtDNA depletion in each of the treated patients, whereas none of 7 patients (and 35 cell specimens) receiving a stavudine + lamivudine + indinavir combination had any significant mtDNA content variations. In 7 patients, mtDNA copy numbers returned to pretreatment levels and/or higher levels without any interruption of the previously mtDNA-depleting antiretroviral drug combination. CONCLUSION: Our assay system allowed the detection of significant changes in the mtDNA content of PBMCs from HIV-1-infected patients taking antiretroviral drugs, as has been reported in the literature with other detection systems. Yet, mtDNA copy numbers regularly diminished during administration of some but not all nucleoside analog-containing combinations. This, plus the occasional finding that depleted mtDNA contents spontaneously increased to baseline levels and/or higher levels during uninterrupted treatment, should raise a note of caution about resorting to the PBMC mtDNA marker for monitoring of antiretroviral drug-related mitochondrial toxicities.