scholarly journals Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors

2019 ◽  
Vol 116 (51) ◽  
pp. 25891-25899 ◽  
Author(s):  
Sean C. Patro ◽  
Leah D. Brandt ◽  
Michael J. Bale ◽  
Elias K. Halvas ◽  
Kevin W. Joseph ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Cell Clone ◽  
Integration Site ◽  
Multiple Displacement Amplification ◽  
Drug Resistant ◽  
Peripheral Blood Mononuclear ◽  
Site Analysis ◽  
T Cell Clone ◽  
Integration Site Analysis ◽  
Hiv 1

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

scholarly journals CpG Methylation Profiles of HIV-1 Pro-Viral DNA in Individuals on ART

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 799
Author(s):  
Valerie F. Boltz ◽  
Cristina Ceriani ◽  
Jason W. Rausch ◽  
Wei Shao ◽  
Michael J. Bale ◽  
...  
Keyword(s):  
Cytosine Methylation ◽  
Mononuclear Cells ◽  
Cell Clone ◽  
Cpg Methylation ◽  
Viral Dna ◽  
Peripheral Blood Mononuclear ◽  
T Cell Clone ◽  
Latent Hiv ◽  
Ltr Promoter ◽  
Hiv 1

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5′ LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5′ LTR promoter.

scholarly journals The use of HIV-1 integration site analysis information in clinical studies aiming at HIV cure

2016 ◽  
Vol 2 (3) ◽  
pp. 175-176
Author(s):  
Maja Kiselinova ◽  
Ward De Spiegelaere ◽  
Linos Vandekerckhove
Keyword(s):  
Clinical Studies ◽  
Integration Site ◽  
Hiv Cure ◽  
Site Analysis ◽  
Integration Site Analysis ◽  
Hiv 1

scholarly journals F18, a Novel Small-Molecule Nonnucleoside Reverse Transcriptase Inhibitor, Inhibits HIV-1 Replication Using Distinct Binding Motifs as Demonstrated by Resistance Selection and Docking Analysis

2011 ◽  
Vol 56 (1) ◽  
pp. 341-351 ◽  
Author(s):  
Xiaofan Lu ◽  
Li Liu ◽  
Xu Zhang ◽  
Terrence Chi Kong Lau ◽  
Stephen Kwok Wing Tsui ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Small Molecule ◽  
Mononuclear Cells ◽  
Binding Motif ◽  
Drug Resistant ◽  
Peripheral Blood Mononuclear ◽  
Docking Analysis ◽  
Hiv 1 ◽  
Calanolide A

ABSTRACTNonnucleoside reverse transcriptase inhibitors (NNRTIs) are one of the key components of antiretroviral therapy drug regimen against human immunodeficiency virus type 1 (HIV-1) replication. We previously described a newly synthesized small molecule, 10-chloromethyl-11-demethyl-12-oxo-calanolide A (F18), a (+)-calanolide A analog, as a novel anti-HIV-1 NNRTI (H. Xue et al., J. Med. Chem. 53:1397–1401, 2010). Here, we further investigated its antiviral range, drug resistance profile, and underlying mechanism of action. F18 consistently displayed potent activity against primary HIV-1 isolates, including various subtypes of group M, circulating recombinant form (CRF) 01_AE, and laboratory-adapted drug-resistant viruses. Moreover, F18 displayed distinct profiles against 17 NNRTI-resistant pseudoviruses, with an excellent potency especially against one of the most prevalent strains with the Y181C mutation (50% effective concentration, 1.0 nM), which was in stark contrast to the extensively used NNRTIs nevirapine and efavirenz. Moreover, we induced F18-resistant viruses byin vitroserial passages and found that the mutation L100I appeared to be the dominant contributor to F18 resistance, further suggesting a binding motif different from that of nevirapine and efavirenz. F18 was nonantagonistic when used in combination with other antiretrovirals against both wild-type and drug-resistant viruses in infected peripheral blood mononuclear cells. Interestingly, F18 displayed a highly synergistic antiviral effect with nevirapine against nevirapine-resistant virus (Y181C). Furthermore,in silicodocking analysis suggested that F18 may bind to the HIV-1 reverse transcriptase differently from other NNRTIs. This study presents F18 as a new potential drug for clinical use and also presents a new mechanism-based design for future NNRTI.

scholarly journals Prediction and Validation of Immunogenic Domains of Pneumococcal Proteins Recognized by Human CD4+T Cells

2019 ◽  
Vol 87 (6) ◽  
Author(s):  
Martijn D. B. van de Garde ◽  
Els van Westen ◽  
Martien C. M. Poelen ◽  
Nynke Y. Rots ◽  
Cécile A. C. M. van Els
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Cell Clone ◽  
Bioinformatics Tool ◽  
Peripheral Blood Mononuclear ◽  
Necrosis Factor Alpha ◽  
T Cell Clone ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACTCD4+T-cell mechanisms are implied in protection against pneumococcal colonization; however, their target antigens and function are not well defined. In contrast to high-throughput protein arrays for serology, basic antigen tools for CD4+T-cell studies are lacking. Here, we evaluate the potential of a bioinformatics tool forin silicoprediction of immunogenicity as a method to reveal domains of pneumococcal proteins targeted by human CD4+T cells. For 100 pneumococcal proteins, CD4+T-cell immunogenicity was predicted based on HLA-DRB1 binding motifs. For 20 potentially CD4+T-cell immunogenic proteins, epitope regions were verified by testing synthetic peptides in T-cell assays using peripheral blood mononuclear cells from healthy adults. Peptide pools of 19 out of 20 proteins evoked T-cell responses. The most frequent responses (detectable in ≥20% of donors tested) were found to SP_0117 (PspA), SP_0468 (putative sortase), SP_0546 (BlpZ), SP_1650 (PsaA), SP_1923 (Ply), SP_2048 (conserved hypothetical protein), SP_2216 (PscB), and SPR_0907 (PhtD). Responding donors had diverging recognition patterns and profiles of signature cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-13 [IL-13], and/or IL-17A) against single-epitope regions. Natural HLA-DR-restricted presentation and recognition of a predicted SP_1923-derived epitope were validated through the isolation of a CD4+T-cell clone producing IFN-γ, TNF-α, and IL-17A in response to the synthetic peptide, whole protein, and heat-inactivated pneumococcus. This proof of principle for a bioinformatics tool to identify pneumococcal protein epitopes targeted by human CD4+T cells provides a peptide-based strategy to study cell-mediated immune mechanisms for the pneumococcal proteome, advancing the development of immunomonitoring assays and targeted vaccine approaches.

scholarly journals Multiple Displacement Amplification Enables Large-Scale Clonal Analysis following Retroviral Gene Therapy

2007 ◽  
Vol 82 (5) ◽  
pp. 2448-2455 ◽  
Author(s):  
S. Bleier ◽  
P. Maier ◽  
H. Allgayer ◽  
F. Wenz ◽  
W. J. Zeller ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Genomic Dna ◽  
Large Scale ◽  
Integration Site ◽  
Multiple Displacement Amplification ◽  
Site Analysis ◽  
Integration Site Analysis ◽  
Reliable Quantification ◽  
Therapy Studies ◽  
First Time

ABSTRACT Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.

scholarly journals Development of Hexadecyloxypropyl Tenofovir (CMX157) for Treatment of Infection Caused by Wild-Type and Nucleoside/Nucleotide-Resistant HIV

2010 ◽  
Vol 54 (7) ◽  
pp. 2901-2909 ◽  
Author(s):  
E. Randall Lanier ◽  
Roger G. Ptak ◽  
Bernhard M. Lampert ◽  
Laurie Keilholz ◽  
Tracy Hartman ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Systemic Exposure ◽  
Target Cells ◽  
Drug Resistant ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Nucleotide Analog ◽  
Antiretroviral Drug ◽  
Hiv 1

ABSTRACT CMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC50s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC50s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitro cytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.

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