Probes for HIV
ACD can configure probes for the various manual and automated assays for HIV for RNAscope Assay, or for Basescope Assay compatible for your species of interest.
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Immunity
2022 Apr 12
Jiang, S;Chan, CN;Rovira-Clavé, X;Chen, H;Bai, Y;Zhu, B;McCaffrey, E;Greenwald, NF;Liu, C;Barlow, GL;Weirather, JL;Oliveria, JP;Nakayama, T;Lee, IT;Matter, MS;Carlisle, AE;Philips, D;Vazquez, G;Mukherjee, N;Busman-Sahay, K;Nekorchuk, M;Terry, M;Younger, S;Bosse, M;Demeter, J;Rodig, SJ;Tzankov, A;Goltsev, Y;McIlwain, DR;Angelo, M;Estes, JD;Nolan, GP;
PMID: 35447093 | DOI: 10.1016/j.immuni.2022.03.020
Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.
Functional impairment of HIV-specific CD8+ T cells precedes aborted spontaneous control of viremia
Immunity
2021 Sep 02
Collins, DR;Urbach, JM;Racenet, ZJ;Arshad, U;Power, KA;Newman, RM;Mylvaganam, GH;Ly, NL;Lian, X;Rull, A;Rassadkina, Y;Yanez, AG;Peluso, MJ;Deeks, SG;Vidal, F;Lichterfeld, M;Yu, XG;Gaiha, GD;Allen, TM;Walker, BD;
PMID: 34496223 | DOI: 10.1016/j.immuni.2021.08.007
Spontaneous control of HIV infection has been repeatedly linked to antiviral CD8+ T cells but is not always permanent. To address mechanisms of durable and aborted control of viremia, we evaluated immunologic and virologic parameters longitudinally among 34 HIV-infected subjects with differential outcomes. Despite sustained recognition of autologous virus, HIV-specific proliferative and cytolytic T cell effector functions became selectively and intrinsically impaired prior to aborted control. Longitudinal transcriptomic profiling of functionally impaired HIV-specific CD8+ T cells revealed altered expression of genes related to activation, cytokine-mediated signaling, and cell cycle regulation, including increased expression of the antiproliferative transcription factor KLF2 but not of genes associated with canonical exhaustion. Lymphoid HIV-specific CD8+ T cells also exhibited poor functionality during aborted control relative to durable control. Our results identify selective functional impairment of HIV-specific CD8+ T cells as prognostic of impending aborted HIV control, with implications for clinical monitoring and immunotherapeutic strategies.
In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization
Frontiers in immunology
2021 Jun 09
Moysi, E;Del Rio Estrada, PM;Torres-Ruiz, F;Reyes-Terán, G;Koup, RA;Petrovas, C;
PMID: 34177929 | DOI: 10.3389/fimmu.2021.683396
CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination.
Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, RT-SHIV RNA, and Collagen in the Mesenteric Lymph Nodes of Nonhuman Primates
Antimicrobial agents and chemotherapy
2021 Mar 29
Scholz, EMB;Mwangi, JN;De la Cruz, G;Nekorchuk, M;Chan, CN;Busman-Sahay, K;Adamson, L;Luciw, P;Fedoriw, Y;Estes, JD;Rosen, EP;Kashuba, ADM;
PMID: 33782003 | DOI: 10.1128/AAC.00019-21
HIV persistence in tissue reservoirs is a major barrier to HIV cure. While antiretrovirals (ARVs) suppress viral replication, antiretroviral therapy (ART) interruption results in rapid rebound viremia that may originate from lymphoid tissues. To understand the relationship between anatomic distribution of ARV exposure and viral expression in lymph nodes, we performed mass spectrometry imaging (MSI) of 6 ARVs, RNAscope in situ hybridization for viral RNA, and immunohistochemistry of collagen in mesenteric lymph nodes from 8 uninfected and 10 reverse transcriptase-simian/human immunodeficiency virus (RT-SHIV) infected rhesus macaque nonhuman primates (NHPs) dosed to steady-state with combination ART. MATLAB-based quantitative imaging analysis was used to evaluate spatial and pharmacologic relationships between these ARVs, viral RNA (both vRNA+ cells and FDC-bound virions) and collagen deposition. Using MSI, 31% of mesenteric lymph node tissue area was not covered by any ARV. Additionally, 28% of FDC-trapped virions and 21% of infected cells were not exposed to any detected ARV. Of the 69% of tissue area that was covered by cumulative ART exposure, nearly 100% of concentrations were greater than in vitro IC50 values; however, 52% of total tissue coverage was from only one ARV, primarily maraviroc. Collagen covered ∼35% of tissue area, but did not influence ARV distribution heterogeneity. Our findings are consistent with our hypothesis that ARV distribution, in addition to total-tissue drug concentration, must be considered when evaluating viral persistence in lymph nodes and other reservoir tissues.
Molecular cell
2022 Dec 15
Plaza-Jennings, AL;Valada, A;O'Shea, C;Iskhakova, M;Hu, B;Javidfar, B;Ben Hutta, G;Lambert, TY;Murray, J;Kassim, B;Chandrasekaran, S;Chen, BK;Morgello, S;Won, H;Akbarian, S;
PMID: 36525955 | DOI: 10.1016/j.molcel.2022.11.016
To explore genome organization and function in the HIV-infected brain, we applied single-nuclei transcriptomics, cell-type-specific chromosomal conformation mapping, and viral integration site sequencing (IS-seq) to frontal cortex from individuals with encephalitis (HIVE) and without (HIV+). Derepressive changes in 3D genomic compartment structures in HIVE microglia were linked to the transcriptional activation of interferon (IFN) signaling and cell migratory pathways, while transcriptional downregulation and repressive compartmentalization of neuronal health and signaling genes occurred in both HIVE and HIV+ microglia. IS-seq recovered 1,221 brain integration sites showing distinct genomic patterns compared with peripheral lymphocytes, with enrichment for sequences newly mobilized into a permissive chromatin environment after infection. Viral transcription occurred in a subset of highly activated microglia comprising 0.33% of all nuclei in HIVE brain. Our findings point to disrupted microglia-neuronal interactions in HIV and link retroviral integration to remodeling of the microglial 3D genome during infection.
PLoS pathogens
2023 Mar 01
Byrnes, SJ;Busman-Sahay, K;Angelovich, TA;Younger, S;Taylor-Brill, S;Nekorchuk, M;Bondoc, S;Dannay, R;Terry, M;Cochrane, CR;Jenkins, TA;Roche, M;Deleage, C;Bosinger, SE;Paiardini, M;Brew, BJ;Estes, JD;Churchill, MJ;
PMID: 36989320 | DOI: 10.1371/journal.ppat.1011290
HIV-associated neurocognitive disorders (HAND) affect ~40% of virally suppressed people with HIV (PWH), however, the precise viral dependent and independent changes to the brain are unclear. Here we characterized the CNS reservoir and immune environment of SIV-infected (SIV+) rhesus macaques during acute (n = 4), chronic (n = 12) or ART-suppressed SIV infection (n = 11). Multiplex immunofluorescence for markers of SIV infection (vRNA/vDNA) and immune activation was performed on frontal cortex and matched colon tissue. SIV+ animals contained detectable viral DNA+ cells that were not reduced in the frontal cortex or the gut by ART, supporting the presence of a stable viral reservoir in these compartments. SIV+ animals had impaired blood brain barrier (BBB) integrity and heightened levels of astrocytes or myeloid cells expressing antiviral, anti-inflammatory or oxidative stress markers which were not abrogated by ART. Neuroinflammation and BBB dysfunction correlated with measures of viremia and immune activation in the gut. Furthermore, SIV-uninfected animals with experimentally induced gut damage and colitis showed a similar immune activation profile in the frontal cortex to those of SIV-infected animals, supporting the role of chronic gut damage as an independent source of neuroinflammation. Together, these findings implicate gut-associated immune activation/damage as a significant contributor to neuroinflammation in ART-suppressed HIV/SIV infection which may drive HAND pathogenesis.
JCI insight
2022 Oct 24
Liu, Z;Julius, P;Kang, G;West, JT;Wood, C;
PMID: 36278485 | DOI: 10.1172/jci.insight.162604
Subtype B HIV-1 reservoirs have been intensively investigated, but reservoirs in other subtypes and how they respond to antiretroviral therapy (ART) is substantially less established. To characterize subtype C HIV-1 reservoirs, we implemented postmortem frozen, as well as formalin fixed paraffin embedded (FFPE) tissue sampling of central nervous system (CNS) and peripheral tissues. HIV-1 LTR, gag, envelope (env) DNA and RNA was quantified using genomic DNA and RNA extracted from frozen tissues. RNAscope was used to localize subtype C HIV-1 DNA and RNA in FFPE tissue. Despite uniform viral load suppression in our cohort, PCR results showed that subtype C HIV-1 proviral copies vary both in magnitude and tissue distribution, with detection primarily in secondary lymphoid tissues. Interestingly, the appendix harbored proviruses in all subjects. Unlike subtype B, subtype C provirus was rarely detectable in the CNS, and there was no detectable HIV-1 RNA. HIV-1 RNA was detected in peripheral lymphoid tissues of 6 out of 8 ART-suppressed cases. In addition to active HIV-1 expression in lymphoid tissues, RNAscope revealed HIV RNA detection in CD4-expressing cells in the appendix, suggesting that this tissue was a previously unreported potential treatment-resistant reservoir for subtype C HIV-1.
Microorganisms
2021 Dec 08
Borrajo, A;Svicher, V;Salpini, R;Pellegrino, M;Aquaro, S;
PMID: 34946138 | DOI: 10.3390/microorganisms9122537
The chronic infection established by the human immunodeficiency virus 1 (HIV-1) produces serious CD4+ T cell immunodeficiency despite the decrease in HIV-1 ribonucleic acid (RNA) levels and the raised life expectancy of people living with HIV-1 (PLWH) through treatment with combined antiretroviral therapies (cART). HIV-1 enters the central nervous system (CNS), where perivascular macrophages and microglia are infected. Serious neurodegenerative symptoms related to HIV-associated neurocognitive disorders (HAND) are produced by infection of the CNS. Despite advances in the treatment of this infection, HAND significantly contribute to morbidity and mortality globally. The pathogenesis and the role of inflammation in HAND are still incompletely understood. Principally, growing evidence shows that the CNS is an anatomical reservoir for viral infection and replication, and that its compartmentalization can trigger the evolution of neurological damage and thus make virus eradication more difficult. In this review, important concepts for understanding HAND and neuropathogenesis as well as the viral proteins involved in the CNS as an anatomical reservoir for HIV infection are discussed. In addition, an overview of the recent advancements towards therapeutic strategies for the treatment of HAND is presented. Further neurological research is needed to address neurodegenerative difficulties in people living with HIV, specifically regarding CNS viral reservoirs and their effects on eradication.
Infection by Diverse HIV-1 Subtypes Leads to Different Elevations in HERV-K Transcriptional Levels in Human T Cell Lines
Frontiers in Microbiology
2021 May 17
Li, X;Guo, Y;Li, H;Huang, X;Pei, Z;Wang, X;Liu, Y;Jia, L;Li, T;Bao, Z;Wang, X;Han, L;Han, J;Li, J;Li, L;
| DOI: 10.3389/fmicb.2021.662573
Human endogenous retroviruses (HERVs) make up ~8% of the human genome, and for millions of years, they have been subject to strict biological regulation. Many HERVs do not participate in normal physiological activities in the body. However, in some pathological conditions, they can be abnormally activated. For example, HIV infection can cause abnormal activation of HERVs, and under different infection conditions, HERV expression may be different. We observed significant differences in HERV-K transcription levels among HIV-1 subtype-infected individuals. The transcriptional levels in the HERV-K gag region were significantly increased in HIV-1 B subtype-infected patients, while the transcriptional levels in the HERV-K pol region were significantly increased in CRF01_AE and CRF07_BC subtype-infected patients. In vitro, the transcriptional levels of HEVR-K were increased 5-fold and 15-fold in MT2 cells transfected with two different HIV-1 strains (B and CRF01_AE, respectively). However, there was no significant difference in transcriptional levels among regions of HERV-K. When MT2 cells were infected with different subtypes of HIV-1 Tat proteins (B, CRF01_AE), which is constructed by lentiviruses, and the transcription levels of HERV-K were increased 4-fold and 2-fold, respectively. Thus, different subtypes of HIV-1 have different effects on HERV-K transcription levels, which may be caused by many factors, not only Tat protein.
bioRxiv : the preprint server for biology
2023 Jan 21
Li, H;McLaurin, KA;Mactutus, CF;Booze, RM;
PMID: 36711456 | DOI: 10.1101/2023.01.20.524942
Microglia, which are productively infected by HIV-1, are critical for brain development and maturation, as well as synaptic plasticity. The pathophysiology of HIV-infected microglia and their role in the pathogenesis of HIV-1-associated neurocognitive and affective alterations, however, remains understudied. Three complementary aims were undertaken to critically address this knowledge gap. First, the predominant cell type expressing HIV-1 mRNA in the dorsolateral prefrontal cortex of postmortem HIV-1 seropositive individuals with HAND was investigated. Utilization of a combined RNAscope multiplex fluorescent and immunostaining assay revealed prominent HIV-1 mRNA in microglia of postmortem HIV-1 seropositive individuals with HAND. Second, measures of microglia proliferation and neuronal damage were evaluated in chimeric HIV (EcoHIV) rats. Eight weeks after EcoHIV innoculation, enhanced microglial proliferation was observed in the medial prefrontal cortex (mPFC) of EcoHIV rats, evidenced by an increased number of cells co-localized with both Iba1+ and Ki67+ relative to control animals. Neuronal damage in EcoHIV infected rats was evidenced by pronounced decreases in both synaptophysin and post synaptic density protein 95 (PSD-95), markers of pre-synaptic and post-synaptic damage, respectively. Third, regression analyses were conducted to evaluate whether microglia proliferation mechanistically underlies neuronal damage in EcoHIV and control animals. Indeed, microglia proliferation accounts for 42-68.6% of the variance in synaptic dysfunction. Collectively, microglia proliferation induced by chronic HIV-1 viral protein exposure may underlie the profound synaptodendritic alterations in HIV-1. Understanding how microglia are involved in the pathogenesis of HAND and HIV-1-associated affective disorders affords a key target for the development of novel therapeutics.
Journal of Virus Eradication
2022 Dec 01
Deleage, C;Fennessey, C;Harper, J;Florea, S;Lipkey, L;Fast, R;Paiardini, M;Lifson, J;Keele, B;
| DOI: 10.1016/j.jve.2022.100170
Background: Most new HIV infections result from sexual interactions with infected but untreated individuals. Semen is the main vector for viral transmission globally, however, little is known regarding the anatomic origin and form of virus in semen. Methods: In this study, we were able to combine numerous new technologies to characterize the virus present in the semen during SIV infection. Six rhesus macaques (RM) were challenged intravenously with barcoded virus SIVmac239M. Semen and blood samples were collected longitudinally for 17 days post-infection with all male genital tract (MGT) and multiple lymphoid tissues collected at necropsy and subjected to quantitative PCR, next generation sequencing of the viral barcode, and tissue analysis (RNAscope, DNAscope and immunophenotyping). Semen was also collected from 6 animals chronically infected with SIVmac251 and in five CD4 depleted animals in acute phase and 2 weeks post ART initiation. Results: Extremely high levels of viral RNA (vRNA) were detected in seminal plasma (up to 10^9cp/ml) as well as comparable levels of cell associated vRNA and vDNA in seminal cells with detection starting as early as 4 days post-infection. RNAscope and immunophenotyping of seminal cells and MGT tissues revealed myeloid cells as the main source of virus (Fig. 1), while CD4+T cells were harboring vRNA in lymphoid tissues. Sequences show evidence of an early compartment between seminal and blood plasma and no difference in the env gene of virus present in semen/MGT and in Lymph Nodes. Finally, multinuclear giant cells harboring vRNA were the only source of virus in semen in chronically infected and in CD4 depleted RM. Moreover, vRNA + myeloid cells were highly present in semen after 2 weeks on ART.
Journal of the International AIDS Society
2022 Apr 01
Rosen, EP;Deleage, C;White, N;Sykes, C;Brands, C;Adamson, L;Luciw, P;Estes, JD;Kashuba, ADM;
PMID: 35441468 | DOI: 10.1002/jia2.25895
HIV reservoirs and infected cells may persist in tissues with low concentrations of antiretrovirals (ARVs). Traditional pharmacology methods cannot assess variability in ARV concentrations within morphologically complex tissues, such as lymph nodes (LNs). We evaluated the distribution of six ARVs into LNs and the proximity of these ARVs to CD4+ T cells and cell-associated RT-SHIV viral RNA.Between December 2014 and April 2017, RT-SHIV infected (SHIV+; N = 6) and healthy (SHIV-; N = 6) male rhesus macaques received two selected four-drug combinations of six ARVs over 10 days to attain steady-state conditions. Serial cryosections of axillary LN were analysed by a multimodal imaging approach that combined mass spectrometry imaging (MSI) for ARV disposition, RNAscope in situ hybridization for viral RNA (vRNA) and immunohistochemistry for CD4+ T cell and collagen expression. Spatial relationships across these four imaging domains were investigated by nearest neighbour search on co-registered images using MATLAB.Through MSI, ARV-dependent, heterogeneous concentrations were observed in different morphological LN regions, such as the follicles and medullary sinuses. After 5-6 weeks of infection, more limited ARV penetration into LN tissue relative to the blood marker heme was found in SHIV+ animals (SHIV+: 0.7 [0.2-1.4] mm; SHIV-: 1.3 [0.5-1.7] mm), suggesting alterations in the microcirculation. However, we found no detectable increase in collagen deposition. Regimen-wide maps of composite ARV distribution indicated that up to 27% of SHIV+ LN tissue area was not exposed to detectable ARVs. Regions associated with B cell follicles had median 1.15 [0.94-2.69] -fold reduction in areas with measurable drug, though differences were only statistically significant for tenofovir (p = 0.03). Median co-localization of drug with CD4+ target cells and vRNA varied widely by ARV (5.1-100%), but nearest neighbour analysis indicated that up to 10% of target cells and cell-associated vRNA were not directly contiguous to at least one drug at concentrations greater than the IC50 value.Our investigation of the spatial distributions of drug, virus and target cells underscores the influence of location and microenvironment within LN, where a small population of T cells may remain vulnerable to infection and low-level viral replication during suppressive ART.
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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 | |
| EnEm | Probe targets exons n and m | |
| En-Em | Probe 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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