Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4$^{+}$ T cell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tissue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and in vitro stimulation, the HIV-1 "Gag and Envelope reactivation co-detection assay" (GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density-based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph-node-homing cells with central memory T cells (T$_{CM}$s) as main players, critical for HIV-1 reservoir eradication

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA : Diagnostic Accuracy, Viral Evolution and Compartmentalization

Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain. We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized. The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making

Das neue Wilhelminenspital: : Vergleich zwischen Ortbeton- und Modulbauweise hinsichtlich Kosten, Terminen und Qualitäten im Krankenhausbau.

Um eine effiziente, leistbare und qualitativ hochwertige wirtschaftliche Gesundheitsversorgung für die Stadt Wien zu schaffen, hat der Wiener Krankenanstaltenverbund das Spitalskonzept 2030 ins Leben gerufen. Bestandteil des Konzepts ist die Errichtung von sieben neuen Zentralspitälern. Eines dieser Spitäler ist das Wilhelminenspital, welches bei laufendem Betrieb des bestehenden Spitals weiter ausgebaut werden soll. Diese Arbeit beschäftigt sich mit dem Vergleich zwischen Ortbeton- und Modulbauweise hinsichtlich Kosten, Terminen und Qualitäten im Krankenhausbau am Beispiel des neuen Wilhelminenspitals. Dazu wurde die folgende Forschungsfrage gestellt: Was sind die Unterschiede bei der Errichtung eines Krankenhauses mittels konventioneller Ortbetonbauweise und Modulbauweise hinsichtlich Kosten, Terminen und Qualitäten? Ist es empfehlenswert, ein Krankenhaus in Modulbauweise zu errichten? Um die Forschungsfrage zu beantworten, wurden die Modulbauweise und die konventionelle Ortbetonbauweise hinsichtlich der Faktoren Kosten, Termine und Qualitäten analysiert. Für die Betrachtung der Kosten wurden die Kosten der Kostengruppen 200 (Erschließung), 300 (Bauwerk – Konstruktion) und 400 (Bauwerk – Ausbau) ermittelt. Für den Faktor Termine wurden die Rahmenterminpläne der jeweiligen Bauweise erstellt und im Folgenden die Abläufe der Planung und Ausführung näher betrachtet. Hinsichtlich des Faktors Qualitäten wurde ein Überblick über die bauphysikalischen Anforderungen (Schallschutz, Brandschutz, Wärmeschutz) erarbeitet und das Thema der Nachhaltigkeit der jeweiligen Bauart betrachtet. Die anschließende Gegenüberstellung der beiden Bauarten führt zu dem Ergebnis, dass vor allem in Bezug auf die Errichtungszeit, Nachhaltigkeit und Minimierung der Störquellen für den laufenden Betrieb die Vorteile der Errichtung eines Spitals in Modulbauweise überwiegen. Auf dieser Grundlage erscheint es empfehlenswert, Krankenhäuser oder Interimskrankenhäuser in Modulbauweise zu errichten.In order to create efficient, affordable and high-quality health care for the City of Vienna, the Vienna Hospital Association has launched the Hospital Concept 2030. The concept includes the construction of seven new central hospitals. One of these hospitals is the Wilhelminenspital, which is to be expanded while the existing hospital continues to operate. This thesis compares in-situ concrete and modular construction methods regarding costs, schedules and qualities in hospital construction using the example of the new Wilhelminenspital. It aims to answer the following research question: What are the differences between the construction of a hospital using conventional in-situ concrete and modular construction methods regarding costs, schedules, and qualities? Is it advisable to build a hospital in modular construction? In order to answer the research question, the modular construction method and the conventional in-situ concrete construction method were analysed regarding costs, schedules and qualities. The costs of cost groups 200 (development), 300 (building - construction), and 400 (building - extension) were determined for the consideration of the costs. Considering the factor schedules, the general schedules of the respective building method were created and in the following the procedures of the planning and execution were analysed in more detail. Considering the factor quality, an overview of the building physics requirements (sound insulation, fire protection, thermal insulation) was compiled and the sustainability of the individual construction type analysed. The comparison of the two types of construction suggests several advantages of modular construction of hospitals, especially regarding the construction time, sustainability, and minimisation of sources of disturbance. On this basis it is recommended to build hospitals or interim hospitals in modular construction

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy, Viral Evolution and Compartmentalization

<div><p>Background</p><p>Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain.</p><p>Methods & Results</p><p>We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No <i>de novo</i> CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.</p><p>Conclusions</p><p>The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.</p></div

Accuracy of Tropism Assays Relative to the Enhanced-Sensitivity Trofile™ Assay.^{^a}

a<p>Values are mean percentages (95% confidence interval of the mean), calculated assuming a binomial distribution of the data.</p>b<p><i>G2P FPR</i>, Geno2Pheno_[coreceptor] false positive rate used for population and 454 sequencing to assign CXCR4 use . The Geno2Pheno_[coreceptor] clonal model was always used.</p>c<p><i>MT-2</i>, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells.</p>d<p><i>PPV</i>, Positive Predictive Value.</p>e<p><i>NPV</i>, Negative Predictive Value.</p>f<p>“Accuracy” is defined as: (True positives + True negatives)/Total.</p

Prevalence of CXCR4-using viruses using different tropism assays and settings.

<p>Bar plot showing the mean and 95% confidence intervals of the prevalence of subjects with CXCR4-using viruses using different tropism assays and settings. The Geno2Pheno_[coreceptor] clinical model was only used in pre-treatment bulk sequences derived from plasma RNA; otherwise, the clonal model was used. ESTA, Enhanced-Sensitivity Trofile™ Assay; FPR, Geno2Pheno_[coreceptor] false positive rate used to assign tropism; MT-2, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells. * p-value<0.05, two-sided exact binomial tst.</p

Selection of a CXCR4-using variant above the 454 sequencing error threshold during persistent viremia suppression in Subject 26.

<p><b><i>Panel A, antiretroviral treatment history, virological and immunological evolution</i></b><b>.</b> Continuous line, HIV-1 RNA levels; dashed line, CD4+ counts; horizontal bars, time period during which a given antiretroviral drug was prescribed. Vertical lines indicate the timepoints when 454 sequencing was performed. LPVr, lopinavir/ritonavir; AZT, zidovudine; ddI, didanosine; RAL, raltegravir. <b><i>Panel B, maximum likelihood nucleotide-based phylogenetic tree</i></b> including V3-loop haplotypes present at a frequency ≥0.6% in the virus population in plasma (triangles), PBMCs before therapy initiation (circles) and PBMCs after persistent viremia suppression (squares). The tree is rooted at the most frequent plasma sequence before antiretroviral treatment initiation. Filled symbols show predicted CXCR4-using viruses; open symbols show predicted CCR5-using viruses. Symbol size increases proportionally to the V3-loop haplotype frequency in the virus population in 10% intervals. Node reliability was tested using 1000 bootstraps; bootstrap values ≥50% are shown. The V3-loop aminoacid sequence translation is shown next to each taxon. Aminoacid changes relative to the predominant sequence in plasma are highlighted in bold and underlined. Gaps correspond to aminoacid indeterminations. A Geno2Pheno _[coreceptor] false positive rate (FPR) equal or lower than 10% was used to define CXCR4 use. The actual false positive rate of each sequence is shown. *Sequence #2 was identical to one detected in 0.04% of PBMC-associated viruses, below the error threshold, before treatment initiation.</p

Longitudinal tropism testing results per subject.^{^a^,^b^,^c}

a<p><i>ESTA</i>, <i>Enhanced-Sensitivity Trofile™ Assay</i>; <i>Pop Seq</i>, population sequencing of the V3-loop; <i>454</i>, 454 sequencing of the V3-loop; <i>MT2</i>, direct co-cultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells. HIV-1 RNA levels are in copies/mL; CD4+ cell counts are in cells/mm³.</p>b<p>Population and 454 sequencing data shown here used the Geno2Pheno_[coreceptor] false positive rate cut-off providing highest accuracy when assigning HIV-1 tropism, i.e.: 20% and 10%, respectively. Based on internal error controls, only V3 forms present in ≥0.6% of viruses were considered for tropism prediction with 454 sequencing.</p>c<p>Tests detecting CXCR4-using HIV are reported as “<i>dual-mixed, DM</i>” for ESTA, <i>“X4”</i> for population sequencing, “<i>percent of X4 viruses”</i> for 454, and “<i>syncytium-inducing, SI</i>” for MT-2 assays; for clarity, viruses only using CCR5 are shown as dashes; NA, tropism test result not available due to lack of amplification.</p

Population structure analysis of plasma and PBMC V3 forms detected by 454 sequencing.^{^a}

a<p>The intracompartment variability (<i>Π</i>) of each sample is measured with the best evolutionary model found with <i>Findmodel</i> (<a href="http://www.hiv.lanl.gov" target="_blank">www.hiv.lanl.gov</a>); it corresponds to the average number of nucleotide differences per site between sequences. Migration events with <i>p-value</i>, and <i>F_ST</i> with <i>p-value</i> are indicated for Slatkin-Madison population structure tests. <i>NA</i> indicates comparisons where the tests were not applicable. <i>NC</i> indicates that variability cannot be calculated because there is only one haplotype.</p>*<p>p-value between 0.05 and 0.01;</p>**<p>p-value<0.01 and 10⁻⁶.</p><p>Statistically significant p-values are colored; the color intensity is proportional to the p-value. Note that a complete dataset was not available for subjects 10 and 13, which were, therefore, not included in this analysis.</p