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Probes for HIV-1

ACD can configure probes for the various manual and automated assays for HIV-1 for RNAscope Assay, or for Basescope Assay compatible for your species of interest.

  • Probes for HIV-1 (0)
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Administration of Panobinostat Is Associated with Increased IL-17A mRNA in the Intestinal Epithelium of HIV-1 Patients

Mediators of Inflammation

2015 Nov 15

Christensen AB, Dige A, Vad-Nielsen J, Brinkmann CR, Bendix M, Østergaard L, Tolstrup M, Søgaard OS, Rasmussen TA, Nyengaard JR, Agnholt J, Denton PW.
PMID: - | DOI: http://dx.doi.org/10.1155/2015/120605

Intestinal CD4+ T cell depletion is rapid and profound during early HIV-1 infection. This leads to a compromised mucosal barrier that prompts chronic systemic inflammation. The preferential loss of intestinal T helper 17 (Th17) cells in HIV-1 disease is a driver of the damage within the mucosal barrier and of disease progression. Thus, understanding the effects of new therapeutic strategies in the intestines has high priority. Histone deacetylase (HDAC) inhibitors (e.g., panobinostat) are actively under investigation as potential latency reversing agents in HIV eradication studies. These drugs have broad effects that go beyond reactivating virus, including modulation of immune pathways. We examined colonic biopsies from ART suppressed HIV-1 infected individuals (clinicaltrials.gov: NCT01680094) for the effects of panobinostat on intestinal T cell activation and on inflammatory cytokine production. We compared biopsy samples that were collected before and during oral panobinostat treatment and observed that panobinostat had a clear biological impact in this anatomical compartment. Specifically, we observed a decrease in CD69+ intestinal lamina propria T cell frequency and increased IL-17A mRNA expression in the intestinal epithelium. These results suggest that panobinostat therapy may influence the restoration of mucosal barrier function in these patients.

Human Interleukin-34 facilitates microglia-like cell differentiation and persistent HIV-1 infection in humanized mice.

Mol Neurodegener.

2019 Mar 05

Mathews S, Branch Woods A, Katano I, Makarov E, Thomas MB, Gendelman HE, Poluektova LY, Ito M, Gorantla S.
PMID: 30832693 | DOI: 10.1186/s13024-019-0311-y

Abstract

BACKGROUND:

Microglia are the principal innate immune defense cells of the centeral nervous system (CNS) and the target of the human immunodeficiency virus type one (HIV-1). A complete understanding of human microglial biology and function requires the cell's presence in a brain microenvironment. Lack of relevant animal models thus far has also precluded studies of HIV-1 infection. Productive viral infection in brain occurs only in human myeloid linage microglia and perivascular macrophages and requires cells present throughout the brain. Once infected, however, microglia become immune competent serving as sources of cellular neurotoxic factors leading to disrupted brain homeostasis and neurodegeneration.

METHODS:

Herein, we created a humanized bone-marrow chimera producing human "microglia like" cells in NOD.Cg-PrkdcscidIl2rgtm1SugTg(CMV-IL34)1/Jic mice. Newborn mice were engrafted intrahepatically with umbilical cord blood derived CD34+ hematopoietic stem progenitor cells (HSPC). After 3 months of stable engraftment, animals were infected with HIV-1ADA, a myeloid-specific tropic viral isolate. Virologic, immune and brain immunohistology were performed on blood, peripheral lymphoid tissues, and brain.

RESULTS:

Human interleukin-34 under the control of the cytomegalovirus promoter inserted in NSG mouse strain drove brain reconstitution of HSPC derived peripheral macrophages into microglial-like cells. These human cells expressed canonical human microglial cell markers that included CD14, CD68, CD163, CD11b, ITGB2, CX3CR1, CSFR1, TREM2 and P2RY12. Prior restriction to HIV-1 infection in the rodent brain rested on an inability to reconstitute human microglia. Thus, the natural emergence of these cells from ingressed peripheral macrophages to the brain could allow, for the first time, the study of a CNS viral reservoir. To this end we monitored HIV-1 infection in a rodent brain. Viral RNA and HIV-1p24 antigens were readily observed in infected brain tissues. Deep RNA sequencing of these infected mice and differential expression analysis revealed human-specific molecular signatures representative of antiviral and neuroinflammatory responses.

CONCLUSIONS:

This humanized microglia mouse reflected human HIV-1 infection in its known principal reservoir and showed the development of disease-specific innate immune inflammatory and neurotoxic responses mirroring what can occur in an infected human brain.

Macrophages but not Astrocytes Harbor HIV DNA in the Brains of HIV-1-Infected Aviremic Individuals on Suppressive Antiretroviral Therapy.

J Neuroimmune Pharmacol.

2018 Sep 07

Ko A, Kang G, Hattler JB, Galadima HI, Zhang J, Li Q, Kim WK.
PMID: 30194646 | DOI: 10.1007/s11481-018-9809-2

The question of whether the human brain is an anatomical site of persistent HIV-1 infection during suppressive antiretroviral therapy (ART) is critical, but remains unanswered. The presence of virus in the brains of HIV patients whose viral load is effectively suppressed would demonstrate not only the potential for CNS to act as an anatomical HIV reservoir, but also the urgent need to understand the factors contributing to persistent HIV behind the blood-brain barrier. Here, we investigated for the first time the presence of cells harboring HIV DNA and RNA in the brains from subjects with undetectable plasma viral load and sustained viral suppression, as identified by the National NeuroAIDS Tissue Consortium. Using new, highly sensitive in situ hybridization techniques, RNAscope and DNAscope, in combination with immunohistochemistry, we were able to detect HIV-1 in the brains of all virally suppressed cases and found that brain macrophages and microglia, but not astrocytes, were the cells harboring HIV DNA in the brain. This study demonstrated that HIV reservoirs persist in brain macrophages/microglia during suppressive ART, which cure/treatment strategies will need to focus on targeting.

Multiplex single-cell visualization of nucleic acids and protein during HIV infection

Nat Commun.

2017 Dec 01

Puray-Chavez M, Tedbury PR, Huber AD, Ukah OB, Yapo V, Liu D, Ji J, Wolf JJ, Engelman AN, Sarafianos SG.
PMID: 29192235 | DOI: 10.1038/s41467-017-01693-z

Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we develop a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV (a) spliced and unspliced RNA, (b) cytoplasmic and nuclear DNA, and (c) Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We also report transcriptional "bursts" of nascent RNA from integrated proviral DNA, and concomitant HIV-1, HIV-2 transcription in co-infected cells. MICDDRP can be used to simultaneously detect multiple viral nucleic acid intermediates, characterize the effects of host factors or drugs on steps of the HIV life cycle, or its reactivation from the latent state, thus facilitating the development of antivirals and latency reactivating agents.

Creation of a long-acting nanoformulated dolutegravir

Nat Commun.

2018 Feb 06

Sillman B, Bade AN, Dash PK, Bhargavan B, Kocher T, Mathews S, Su H, Kanmogne GD, Poluektova LY, Gorantla S, McMillan J, Gautam N, Alnouti Y, Edagwa B, Gendelman HE.
PMID: 29402886 | DOI: 10.1038/s41467-018-02885-x

Potent antiretroviral activities and a barrier to viral resistance characterize the human immunodeficiency virus type one (HIV-1) integrase strand transfer inhibitor dolutegravir (DTG). Herein, a long-acting parenteral DTG was created through chemical modification to improve treatment outcomes. A hydrophobic and lipophilic modified DTG prodrug is encapsulated into poloxamer nanoformulations (NMDTG) and characterized by size, shape, polydispersity, and stability. Retained intracytoplasmic NMDTG particles release drug from macrophages and attenuate viral replication and spread of virus to CD4+ T cells. Pharmacokinetic tests in Balb/cJ mice show blood DTG levels at, or above, its inhibitory concentration90 of 64 ng/mL for 56 days, and tissue DTG levels for 28 days. NMDTG protects humanized mice from parenteral challenge of the HIV-1ADA strain for two weeks. These results are a first step towards producing a long-acting DTG for human use by affecting drug apparent half-life, cell and tissue drug penetration, and antiretroviral potency.

Defining HIV and SIV Reservoirs in Lymphoid Tissues

Pathogens and Immunity

2016 Jun 14

Deleage C, Wietgrefe SW, Del Prete G, Morcock DR, Hao XP, Anderson JL, Perkey K, Piatak M, Bess J, Reilly C, McCune JM, Haase AT, Lifson JD, Schacker TW, Estes JD.
PMID: - | DOI: 10.20411/pai.v1i1.100

A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles (“RNAscope”), vDNA+ cells (“DNAscope”) and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situhybridization. Highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.

 
CTLA-4+PD-1− Memory CD4+ T Cells Critically Contribute to Viral Persistence in Antiretroviral Therapy-Suppressed, SIV-Infected Rhesus Macaques

Immunity.

2017 Oct 17

McGary CS, Deleage C, Harper J, Micci L, Ribeiro SP, Paganini S, Kuri-Cervantes L, Benne C, Ryan ES, Balderas R, Jean S, Easley K, Marconi V, Silvestri G, Estes JD, Sekaly RP, Paiardini M.
PMID: 29045906 | DOI: 10.1016/j.immuni.2017.09.018

Antiretroviral therapy (ART) suppresses viral replication in HIV-infected individuals but does not eliminate the reservoir of latently infected cells. Recent work identified PD-1+ follicular helper T (Tfh) cells as an important cellular compartment for viral persistence. Here, using ART-treated, SIV-infected rhesus macaques, we show that CTLA-4+PD-1- memory CD4+ T cells, which share phenotypic markers with regulatory T cells, were enriched in SIV DNA in blood, lymph nodes (LN), spleen, and gut, and contained replication-competent and infectious virus. In contrast to PD-1+ Tfh cells, SIV-enriched CTLA-4+PD-1- CD4+ T cells were found outside the B cell follicle of the LN, predicted the size of the persistent viral reservoir during ART, and significantly increased their contribution to the SIV reservoir with prolonged ART-mediated viral suppression. We have shown that CTLA-4+PD-1- memory CD4+ T cells are a previously unrecognized component of the SIV and HIV reservoir that should be therapeutically targeted for a functional HIV-1 cure.

Tracking the Luminal Exposure and Lymphatic Drainage Pathways of Intravaginal and Intrarectal Inocula Used in Nonhuman Primate Models of HIV Transmission.

PLoS One. 2014 Mar 25;9(3):e92830.

Smedley J, Turkbey B, Bernardo ML, Del Prete GQ, Estes JD, Griffiths GL, Kobayashi H, Choyke PL, Lifson JD, Keele BF.
PMID: 24667371 | DOI: 10.1371/journal.pone.0092830.

Over 80% of sexual HIV-1 transmissions originate from a single viral variant, but the underlying basis for this transmission bottleneck remains to be elucidated. Nonhuman primate models of mucosal virus transmission allow opportunities to gain insight into the basis of this mucosal bottleneck. We used simulated inocula consisting of either non-infectious vital dye or contrast dye with non-invasive magnetic resonance imaging (MRI) to visualize mucosal exposure and passive lymphatic drainage patterns following vaginal and rectal exposures in Indian origin rhesus macaques. Results revealed a limited overall distance of dye coverage from the anal verge following 1 ml (n  = 8) intrarectally administered, which greatly increased with a 3 ml (n = 8) volume. Intravaginal dye exposure using 2 ml revealed complete coverage of the mucosa of the vagina and ectocervix, however dye was not detectable in the endocervix, uterus, fallopian tubes or ovaries in nuliparous sexually mature rhesus macaques (n = 9). In addition, following submucosal and intranodal injections of vital dye or MRI contrast dye in the rectum (n = 9), or distal and proximal vagina (n = 4), the lymphatic drainage pathways were identified as first the internal then common iliac chain followed by para-aortic lymph nodes. Drainage from the distal descending colon (n = 8) was via the para-colonic lymph nodes followed by the inferior mesenteric and para-aortic lymph nodes. Analysis after vaginal challenge with infectious SIVmac239 followed by euthanasia at day 3 revealed a pattern of viral dissemination consistent with the imaging results. These results provide insights into potential patterns of viral dissemination that can help guide efforts to better elucidate the earliest events of virus transmission and potential intervention strategies.
Reactivation of SIV reservoirs in the brain of virally suppressed macaques.

AIDS.

2016 Sep 20

Gama L, Abreu CM, Shirk EN, Price SL, Li M, Laird GM, Pate KA, Wietgrefe SW, O'connor SL, Pianowski L, Haase AT, Van Lint C, Siliciano RF, Clements JE, Authors D; LRA-SIV Study Group.
PMID: 27662554 | DOI: 10.1097/QAD.0000000000001267

Abstract

OBJECTIVE:

Resting CD4+ T cells have been recognized as the major cell reservoir of latent HIV-1 during antiretroviral therapy (ART). Using an SIV/macaque model for AIDS and HIV-related neurocognitive disorders we assessed the contribution of the brain to viral latency and reactivation.

DESIGN:

Pigtailed macaques were dual inoculated with SIVDeltaB670 and SIV17E-Fr and treated with an efficacious CNS-penetrant ART. After 500 days of viral suppression animals were treated with two cycles of latency reversing agents (LRAs) and increases in viral transcripts were examined.

METHODS:

Longitudinal plasma and CSF viral loads were analyzed by quantitative and digital droplet PCR. After necropsy, viral transcripts in organs were analyzed by PCR, in situ hybridization (ISH), and phylogenetic genotyping based on env V1 loop sequences. Markers for neuronal damage and CSF activation were measured by ELISA.

RESULTS:

Increases in activation markers and plasma and CSF viral loads were observed in one animal treated with LRAs, despite ongoing ART. SIV transcripts were identified in occipital cortex macrophages by ISH and CD68+ staining. The most abundant SIV genotype in CSF was unique and expanded independent from viruses found in the periphery.

CONCLUSION:

The CNS harbors latent SIV genomes after long-term viral suppression by ART indicating that the brain represents a potential viral reservoir and should be seriously considered during AIDS cure strategies.

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Description
sense
Example: Hs-LAG3-sense
Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron#
Example: Mm-Htt-intron2
Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan
Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)
A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp
Example: Hs-PDGFB-No-XMm
Does not cross detect with the species (Sp)
XSp
Example: Rn-Pde9a-XMm
designed to cross detect with the species (Sp)
O#
Example: Mm-Islr-O1
Alternative design targeting different regions of the same transcript or isoforms
CDS
Example: Hs-SLC31A-CDS
Probe targets the protein-coding sequence only
EnEmProbe targets exons n and m
En-EmProbe targets region from exon n to exon m
Retired Nomenclature
tvn
Example: Hs-LEPR-tv1
Designed to target transcript variant n
ORF
Example: Hs-ACVRL1-ORF
Probe targets open reading frame
UTR
Example: Hs-HTT-UTR-C3
Probe targets the untranslated region (non-protein-coding region) only
5UTR
Example: Hs-GNRHR-5UTR
Probe targets the 5' untranslated region only
3UTR
Example: Rn-Npy1r-3UTR
Probe targets the 3' untranslated region only
Pan
Example: Pool
A mixture of multiple probe sets targeting multiple genes or transcripts

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