scholarly journals Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA

2020 ◽  
Vol 117 (31) ◽  
pp. 18692-18700 ◽  
Author(s):  
Francesco R. Simonetti ◽  
Jennifer A. White ◽  
Camille Tumiotto ◽  
Kristen D. Ritter ◽  
Mian Cai ◽  
...  
Keyword(s):  
T Cells ◽  
False Negative ◽  
Basic Research ◽  
The United States ◽  
Quantitative Evidence ◽  
Dna Assay ◽  
Proviral Dna ◽  
Negative Results ◽  
Hiv 1

A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1+adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/106CD4+T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/106CD4+T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.

DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

2020 ◽  
Author(s):  
M.A. Tyumentseva ◽  
◽  
A.I. Tyumentsev ◽  
V.G. Akimkin ◽  
◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Health Monitoring ◽  
Biological Samples ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Low Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

Aspiration Revisited: Prospective Evaluation of a Physiologically Pressurized Model With Animal Correlation and Broader Applicability to Filler Complications

2021 ◽  
Author(s):  
Hyoung-Jin Moon ◽  
Won Lee ◽  
Ji-Soo Kim ◽  
Eun-Jung Yang ◽  
Hema Sundaram
Keyword(s):  
Normal Saline ◽  
False Negative ◽  
Vascular Complications ◽  
Filler Injection ◽  
Negative Results ◽  
Needle Position ◽  
False Negative Results ◽  
In Vivo Testing

Abstract Background Aspiration testing before filler injection is controversial. Some believe that aspiration can help prevent inadvertent intravascular injection, while others cite false-negative results and question its value given that the needle position always changes somewhat during injection procedures. Objectives To test the relation of false-negative results to the viscosity of the material within the needle lumen and determine whether a less viscous material within the needle lumen could decrease the incidence of false-negative results. Methods In vitro aspiration tests were performed using 30-G and 27-G needle gauges, two cross-linked hyaluronic acid fillers, normal saline bags pressurized at 140 and 10 mmHg to mimic human arterial and venous pressures, and three needle lumen conditions (normal saline, air, and filler). Testing was repeated three times under each study condition (72 tests in total). For in vivo correlation, aspiration tests were performed on femoral arteries and central auricular veins in three rabbits (4–5 aspirations per site, 48 tests in total). Results In vitro and in vivo testing using 30-G needles containing filler both showed false-negative results on aspiration testing. In vitro and in vivo testing using needles containing saline or air showed positive findings. Conclusions False-negative results from aspiration testing may be reduced by pre-filling the needle lumen with saline rather than a filler. The pressurized system may help overcome challenges of animal models with intravascular pressures significantly different from those of humans. The adaptability of this system to mimic various vessel pressures may facilitate physiologically relevant studies of vascular complications.

scholarly journals G2-S16 Polyanionic Carbosilane Dendrimer Can Reduce HIV-1 Reservoir Formation by Inhibiting Macrophage Cell to Cell Transmission

2021 ◽  
Vol 22 (16) ◽  
pp. 8366
Author(s):  
Ignacio Relaño-Rodríguez ◽  
María de la Sierra Espinar-Buitrago ◽  
Vanessa Martín-Cañadilla ◽  
Rafael Gómez-Ramírez ◽  
María Ángeles Muñoz-Fernández
Keyword(s):  
T Cells ◽  
Developed Countries ◽  
Main Role ◽  
Viral Particles ◽  
Carbosilane Dendrimer ◽  
Science Community ◽  
New Compounds ◽  
Hiv 1

Human immunodeficiency virus (HIV-1) is still a major problem, not only in developing countries but is also re-emerging in several developed countries, thus the development of new compounds able to inhibit the virus, either for prophylaxis or treatment, is still needed. Nanotechnology has provided the science community with several new tools for biomedical applications. G2-S16 is a polyanionic carbosilane dendrimer capable of inhibiting HIV-1 in vitro and in vivo by interacting directly with viral particles. One of the main barriers for HIV-1 eradication is the reservoirs created in primoinfection. These reservoirs, mainly in T cells, are untargetable by actual drugs or immune system. Thus, one approach is inhibiting HIV-1 from reaching these reservoir cells. In this context, macrophages play a main role as they can deliver viral particles to T cells establishing reservoirs. We showed that G2-S16 dendrimer is capable of inhibiting the infection from infected macrophages to healthy T CD4/CD8 lymphocytes by eliminating HIV-1 infectivity inside macrophages, so they are not able to carry infectious particles to other body locations, thus preventing the reservoirs from forming.

scholarly journals CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection

2021 ◽  
Vol 22 (2) ◽  
pp. 912
Author(s):  
Nabila Seddiki ◽  
John Zaunders ◽  
Chan Phetsouphanh ◽  
Vedran Brezar ◽  
Yin Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Proportion ◽  
Long Term Memory ◽  
Ki 67 ◽  
Memory Cd4 T Cells ◽  
Hiv 1

HIV-1 infection rapidly leads to a loss of the proliferative response of memory CD4+ T lymphocytes, when cultured with recall antigens. We report here that CD73 expression defines a subset of resting memory CD4+ T cells in peripheral blood, which highly express the α-chain of the IL-7 receptor (CD127), but not CD38 or Ki-67, yet are highly proliferative in response to mitogen and recall antigens, and to IL-7, in vitro. These cells also preferentially express CCR5 and produce IL-2. We reasoned that CD73+ memory CD4+ T cells decrease very early in HIV-1 infection. Indeed, CD73+ memory CD4+ T cells comprised a median of 7.5% (interquartile range: 4.5–10.4%) of CD4+ T cells in peripheral blood from healthy adults, but were decreased in primary HIV-1 infection to a median of 3.7% (IQR: 2.6–6.4%; p = 0.002); and in chronic HIV-1 infection to 1.9% (IQR: 1.1–3%; p < 0.0001), and were not restored by antiretroviral therapy. Moreover, we found that a significant proportion of CD73+ memory CD4+ T cells were skewed to a gut-homing phenotype, expressing integrins α4 and β7, CXCR3, CCR6, CD161 and CD26. Accordingly, 20% of CD4+ T cells present in gut biopsies were CD73+. In HIV+ subjects, purified CD73+ resting memory CD4+ T cells in PBMC were infected with HIV-1 DNA, determined by real-time PCR, to the same level as for purified CD73-negative CD4+ T cells, both in untreated and treated subjects. Therefore, the proliferative CD73+ subset of memory CD4+ T cells is disproportionately reduced in HIV-1 infection, but, unexpectedly, their IL-7 dependent long-term resting phenotype suggests that residual infected cells in this subset may contribute significantly to the very long-lived HIV proviral DNA reservoir in treated subjects.

scholarly journals An anti-CD45RO immunotoxin eliminates T cells latently infected with HIV-1 in vitro

1999 ◽  
Vol 96 (20) ◽  
pp. 11482-11485 ◽  
Author(s):  
C. McCoig ◽  
G. Van Dyke ◽  
C.-S. Chou ◽  
L. J. Picker ◽  
O. Ramilo ◽  
...  
Keyword(s):  
T Cells ◽  
Latently Infected ◽  
Hiv 1

scholarly journals Infection of CD8+CD45RO+ Memory T-Cells by HIV-1 and Their Proliferative Response

2008 ◽  
Vol 2 (1) ◽  
pp. 43-57 ◽  
Author(s):  
Naveed Gulzar ◽  
Sowyma Balasubramanian ◽  
Greg Harris ◽  
Jaime Sanchez-Dardon ◽  
Karen F.T. Copeland
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Proliferative Response ◽  
Cell Infection ◽  
Cellular Target ◽  
Potential Reservoir ◽  
Infected Cells ◽  
Hiv 1

CD8+ T-cells are involved in controlling HIV-1 infection by eliminating infected cells and secreting soluble factors that inhibit viral replication. To investigate the mechanism and significance of infection of CD8+ T-cells by HIV-1in vitro, we examined the susceptibility of these cells and their subsets to infection. CD8+ T-cells supported greater levels of replication with T-cell tropic strains of HIV-1, though viral production was lower than that observed in CD4+ T-cells. CD8+ T-cell infection was found to be productive through ELISA, RT-PCR and flow cytometric analyses. In addition, the CD8+CD45RO+ memory T-cell population supported higher levels of HIV-1 replication than CD8+CD45RA+ naïve T-cells. However, infection of CD8+CD45RO+ T-cells did not affect their proliferative response to the majority of mitogens tested. We conclude, with numerous lines of evidence detecting and measuring infection of CD8+ T-cells and their subsets, that this cellular target and potential reservoir may be central to HIV-1 pathogenesis.

scholarly journals Preferential and persistent impact of acute HIV-1 infection on CD4+ iNKT cells in colonic mucosa

2021 ◽  
Vol 118 (46) ◽  
pp. e2104721118
Author(s):  
Dominic Paquin-Proulx ◽  
Kerri G. Lal ◽  
Yuwadee Phuang-Ngern ◽  
Matthew Creegan ◽  
Andrey Tokarev ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Colonic Mucosa ◽  
Inkt Cells ◽  
Elevated Expression ◽  
Acute Hiv ◽  
The Impact ◽  
Hiv 1

Acute HIV-1 infection (AHI) results in the widespread depletion of CD4+ T cells in peripheral blood and gut mucosal tissue. However, the impact on the predominantly CD4+ immunoregulatory invariant natural killer T (iNKT) cells during AHI remains unknown. Here, iNKT cells from peripheral blood and colonic mucosa were investigated during treated and untreated AHI. iNKT cells in blood were activated and rapidly depleted in untreated AHI. At the time of peak HIV-1 viral load, these cells showed the elevated expression of cell death–associated transcripts compared to preinfection. Residual peripheral iNKT cells suffered a diminished responsiveness to in vitro stimulation early into chronic infection. Additionally, HIV-1 DNA, as well as spliced and unspliced viral RNA, were detected in iNKT cells isolated from blood, indicating the active infection of these cells in vivo. The loss of iNKT cells occurred from Fiebig stage III in the colonic mucosa, and these cells were not restored to normal levels after initiation of ART during AHI. CD4+ iNKT cells were depleted faster and more profoundly than conventional CD4+ T cells, and the preferential infection of CD4+ iNKT cells over conventional CD4+ T cells was confirmed by in vitro infection experiments. In vitro data also provided evidence of latent infection in iNKT cells. Strikingly, preinfection levels of peripheral blood CD4+ iNKT cells correlated directly with the peak HIV-1 load. These findings support a model in which iNKT cells are early targets for HIV-1 infection, driving their rapid loss from circulation and colonic mucosa.

scholarly journals Evaluation of HIV-1 latency reversal and antibody-dependent viral clearance by quantification of singly spliced HIV-1 vpu/env mRNA

2021 ◽  
Author(s):  
Hongbo Gao ◽  
Ayşe N. Ozantürk ◽  
Qiankun Wang ◽  
Gray H. Harlan ◽  
Aaron J. Schmitz ◽  
...  
Keyword(s):  
T Cells ◽  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Viral Envelope ◽  
Latent Reservoir ◽  
Broadly Neutralizing Antibodies ◽  
Major Barrier ◽  
Hiv 1 ◽  
Reversing Agents

The latent reservoir of HIV-1 is a major barrier for viral eradication. Potent HIV-1 broadly neutralizing antibodies (bNabs) have been used to prevent and treat HIV-1 infections in animal models and clinical trials. Combination of bNabs and latency-reversing agents (LRAs) is considered a promising approach for HIV-1 eradication. PCR-based assays that can rapidly and specifically measure singly spliced HIV-1 vpu/env mRNA are needed to evaluate the induction of the viral envelope production at the transcription level and bNab-mediated reservoir clearance. Here we reported a PCR-based method to accurately quantify the production of intracellular HIV-1 vpu/env mRNA. With the vpu/env assay, we determined the LRA combinations that could effectively induce vpu/env mRNA production in CD4+ T cells from ART-treated individuals. None of the tested LRAs were effective alone. A comparison between the quantitative viral outgrowth assay (Q-VOA) and the vpu/env assay showed that vpu/env mRNA production was closely associated with the reactivation of replication-competent HIV-1, suggesting that vpu/env mRNA was mainly produced by intact viruses. Finally, antibody-mediated in vitro killing in HIV-1-infected humanized mice demonstrated that the vpu/env assay could be used to measure the reduction of infected cells in tissues and was more accurate than the commonly used gag-based PCR assay which measured unspliced viral genomic RNA. In conclusion, the vpu/env assay allows convenient and accurate assessment of HIV-1 latency reversal and bNab-mediated therapeutic strategies. Importance HIV-1 persists in individuals on antiretroviral therapy (ART) due to the long-lived cellular reservoirs that contain dormant viruses. Recent discoveries of HIV-1-specific broadly neutralizing antibodies (bNabs) targeting HIV-1 Env protein rekindled the interest in antibody-mediated elimination of latent HIV-1. Latency-reversing agents (LRAs) together with HIV-1 bNabs is a possible strategy to clear residual viral reservoirs, which makes the evaluation of HIV-1 Env expression upon LRA treatment critical. We developed a PCR-based assay to quantify the production of intracellular HIV-1 vpu/env mRNA. Using patient CD4+ T cells, we found that induction of HIV-1 vpu/env mRNA required a combination of different LRAs. Using in vitro, ex vivo and humanized mouse models, we showed that the vpu/env assay could be used to measure antibody efficacy in clearing HIV-1 infection. These results suggest that the vpu/env assay can accurately evaluate HIV-1 reactivation and bNab-based therapeutic interventions.

Synthesis, Characterization and anti-HIV and Antitumor Activities of New Coumarin Derivatives

2008 ◽  
Vol 63 (1) ◽  
pp. 83-89 ◽  
Author(s):  
Yaseen A. Al-Soud ◽  
Haitham H. Al-Sa’doni ◽  
Houssain A. S. Amajaour ◽  
Kifah S. M. Salih ◽  
Mohammad S. Mubarakb ◽  
...  
Keyword(s):  
T Cells ◽  
In Vitro Cytotoxicity ◽  
Tumor Cell Lines ◽  
Coumarin Derivatives ◽  
Antitumor Activities ◽  
Reverse Transcriptase Inhibitors ◽  
Human T Cells ◽  
Anti Hiv ◽  
Hiv 1

A new series of coumarin and benzofuran derivatives were synthesized as potential non-nucleoside reverse transcriptase inhibitors (NNRTIs) by reacting, separately, 4-bromomethylcoumarins, their sulphonyl chlorides, and ethyl 3-(bromomethyl)-6-methoxy-1-benzofuran-2-carboxylate with different imidazoles and their benzo analogs. The antiviral (HIV-1, HIV-2) properties of the newly synthesized compounds were investigated in vitro and all compounds were found to be inactive, except 10 which showed inhibition of HIV-2 with EC50 > 0.51 μgmL−1. The in vitro cytotoxicity of 17 and 19 was assayed against a panel of tumor cell lines consisting of CD4 human T-cells.

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