Probes for INS

Despite the success of antiretroviral therapy (ART) in transforming HIV disease into a chronic infection, people living with HIV (PLWH) remain at risk for various non-AIDS inflammatory comorbidities. Risk of non-AIDS comorbidities is associated with gut dysbiosis, epithelial gut damage and subsequent microbial translocation, and increased activation of both circulating CD4+ and CD8+ T-cells. Therefore, in addition to ART, novel gut microbiota-modulating therapies could aid in reducing inflammation and immune activation, gut damage, and microbial translocation. Among various gut-modulation strategies under investigation, the Amazonian fruit Camu Camu (CC) presents itself as a prebiotic candidate based on its anti-inflammatory and antioxidant properties in animal models and tobacco smokers.A total of 22 PLWH on ART for more than 2 years, with a viral load <50 copies/mL, a CD4 +count >200 and a CD4+/CD8 +ratio <1 (suggesting increased inflammation and risk for non-AIDS comorbidities), will be recruited in a single arm, non-randomised, interventional pilot trial. We will assess tolerance and effect of supplementation with CC in ART-treated PLWH on reducing gut damage, microbial translocation, inflammation and HIV latent reservoir by various assays.The Canadian Institutes of Health Research (CIHR)/Canadian HIV Trials Network (CTN) pilot trial protocol CTNPT032 was approved by the Natural and Non-prescription Health Products Directorate of Health Canada and the research ethics board of the McGill university Health Centre committee (number 2020-5903). Results will be made available as free access through publications in peer-reviewed journals and through the CIHR/CTN website.NCT04058392.

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.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

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.

HIV persistence in the CNS despite antiretroviral therapy may cause neurological disorders and poses a critical challenge for HIV cure. Understanding the pathobiology of HIV-infected microglia, the main viral CNS reservoir, is imperative. Here, we provide a comprehensive comparison of human microglial culture models: cultured primary microglia (pMG), microglial cell lines, monocyte-derived microglia (MDMi), stem cell-derived microglia (iPSC-MG), and microglia grown in 3D cerebral organoids (oMG) as potential model systems to advance HIV research on microglia. Functional characterization revealed phagocytic capabilities and responsiveness to LPS across all models. Microglial transcriptome profiles of uncultured pMG showed the highest similarity to cultured pMG and oMG, followed by iPSC-MG and then MDMi. Direct comparison of HIV infection showed a striking difference, with high levels of viral replication in cultured pMG and MDMi and relatively low levels in oMG resembling HIV infection observed in post-mortem biopsies, while the SV40 and HMC3 cell lines did not support HIV infection. Altogether, based on transcriptional similarities to uncultured pMG and susceptibility to HIV infection, MDMi may serve as a first screening tool, whereas oMG, cultured pMG, and iPSC-MG provide more representative microglial culture models for HIV research. The use of current human microglial cell lines (SV40, HMC3) is not recommended.

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

The HIV-1 often evades a robust antiretroviral-mediated immune response, leading to persistent infection within anatomically privileged sites including the CNS. Continuous low-level infection occurs in the presence of effective antiretroviral therapy (ART) in CD4+ T cells and mononuclear phagocytes (MP; monocytes, macrophages, microglia, and dendritic cells). Within the CNS, productive viral infection is found exclusively in microglia and meningeal, perivascular, and choroidal macrophages. MPs serve as the principal viral CNS reservoir. Animal models have been developed to recapitulate natural human HIV-1 infection. These include nonhuman primates, humanized mice, EcoHIV, and transgenic rodent models. These models have been used to study disease pathobiology, antiretroviral and immune modulatory agents, viral reservoirs, and eradication strategies. However, each of these models are limited to specific component(s) of human disease. Indeed, HIV-1 species specificity must drive therapeutic and cure studies. These have been studied in several model systems reflective of latent infections, specifically in MP (myeloid, monocyte, macrophages, microglia, and histiocyte cell) populations. Therefore, additional small animal models that allow productive viral replication to enable viral carriage into the brain and the virus-susceptible MPs are needed. To this end, this review serves to outline animal models currently available to study myeloid brain reservoirs and highlight areas that are lacking and require future research to more effectively study disease-specific events that could be useful for viral eradication studies both in and outside the CNS.

The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.

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.

The relationship between spatiotemporal distribution of HIV-1 proviruses and their transcriptional activity is not well understood. To elucidate the intranuclear positions of transcriptionally active HIV-1 proviruses, we utilized an RNA fluorescence in situ hybridization assay and RNA stem loops that bind to fluorescently labeled bacterial protein (Bgl-mCherry) to specifically detect HIV-1 transcription sites. Initially, transcriptionally active wild-type proviruses were located closer to the nuclear envelope (NE) than expected by random chance in HeLa (∼1.4 μm) and CEM-SS T cells (∼0.9 μm). Disrupting interactions between HIV-1 capsid and host cleavage and polyadenylation specificity factor (CPSF6) resulted in localization of proviruses to lamina-associated domains (LADs) adjacent to the NE in HeLa cells (∼0.9 - 1.0 μm); however, in CEM-SS T cells, there was little or no shift toward the NE (∼0.9 μm), indicating cell-type differences in the locations of transcriptionally active proviruses. The distance from the NE was not correlated with transcriptional activity, and transcriptionally active proviruses were randomly distributed throughout the HeLa cell after several cell divisions, indicating that the intranuclear locations of the chromosomal sites of integration are dynamic. After nuclear import HIV-1 cores colocalized with nuclear speckles, nuclear domains enriched in pre-mRNA splicing factors, but transcriptionally active proviruses detected 20 h after infection were mostly located outside but near nuclear speckles, suggesting a dynamic relationship between the speckles and integration sites. Overall, these studies establish that the nuclear distribution of HIV-1 proviruses is dynamic and the distance between HIV-1 proviruses and the NE does not correlate with transcriptional activity. IMPORTANCE HIV-1 integrates its genomic DNA into the chromosomes of the infected cell, but how it selects the site of integration and the impact of their location in the 3-dimensional nuclear space is not well understood. Here, we examined the nuclear locations of proviruses 1 and 5 days after infection and found that integration sites are first located near the nuclear envelope but become randomly distributed throughout the nucleus after a few cell divisions, indicating that the locations of the chromosomal sites of integration that harbor transcriptionally active proviruses are dynamic. We also found that the distance from the nuclear envelope to the integration site is cell-type dependent and does not correlate with proviral transcription activity. Finally, we observed that HIV-1 cores were localized to nuclear speckles shortly after nuclear import, but transcriptionally active proviruses were located adjacent to nuclear speckles. Overall, these studies provide insights into HIV-1 integration site selection and their effect on transcription activities.

Current antiretroviral treatment fails to cure HIV-1 infection since latent provirus resides in long-lived cellular reservoirs, rebounding whenever therapy is discontinued. The molecular mechanisms underlying HIV-1 latency are complex where the possible link between integration and transcription is poorly understood. HIV-1 integration is targeted toward active chromatin by the direct interaction with a host protein, lens epithelium-derived growth factor (LEDGF/p75). LEDGINs are small-molecule inhibitors of the LEDGF/p75-integrase (IN) interaction that effectively inhibit and retarget HIV-1 integration out of preferred integration sites, resulting in residual provirus that is more latent. Here, we describe a single-cell branched DNA imaging method for simultaneous detection of viral DNA and RNA. We investigated how treatment with LEDGINs affects the location, transcription, and reactivation of HIV-1 in both cell lines and primary cells. This approach demonstrated that LEDGIN-mediated retargeting hampered the baseline transcriptional state and the transcriptional reactivation of the provirus, evidenced by the reduction in viral RNA expression per residual copy. Moreover, treatment of primary cells with LEDGINs induced an enrichment of provirus in deep latency. These results corroborate the impact of integration site selection for the HIV-1 transcriptional state and support block-and-lock functional cure strategies in which the latent reservoir is permanently silenced after retargeting. IMPORTANCE A longstanding question exists on the impact of the HIV-1 integration site on viral gene expression. This unsolved question has significant implications for the search toward an HIV-1 cure, as eradication strategies set up to reactivate and eliminate HIV-1 depend on the site where the provirus is integrated. The main determinant for integration site selection is the interaction of the HIV-1 integrase (IN) and the host chromatin targeting factor, LEDGF/p75. LEDGINs are small-molecule inhibitors of the LEDGF/p75-IN interaction that inhibit and retarget HIV-1 integration out of preferred integration sites. Using both LEDGINs and branched DNA (bDNA) imaging, we now investigated, in much detail, the impact of integration site selection on the three-dimensional location of the provirus, HIV-1 transcription, and reactivation. Our results provide evidence for a "block-and-lock" functional cure strategy that aims to permanently silence HIV-1 by LEDGIN-mediated retargeting to sites that are less susceptible to reactivation after treatment interruption.

Background: Early after primary infection, HIV reservoirs are established within multiple tissues, including the brain. As these viral reservoirs are not targeted by antiretroviral therapy (cART), we require robust methods of detection, quantification, and characterization of these viral reservoirs in human tissues. Our recent work developed a multi-component imaging methodology that characterizes and quantifies viral reservoirs within the brain. Methods: The imaging methodology demonstrated utilizes the simultaneous staining of brain tissue from HIV-infected donors using DNAscope, RNAscope, and antibodies for HIV-DNA, HIV-mRNA, and either viral or host proteins, respectively. The panel of patients included in these analyses varied in cART regimen, viral load, years living with HIV, and neurocognitive status, all contrasted to age-matched tissues from uninfected patients. Results: Our group demonstrated that cART is sufficient to reduce the size of the viral reservoirs within the brains of HIV patients. We also found that about half of the cells positive for HIV-DNA expressed HIV-mRNA, and only about one-third expressed viral proteins. HIV proteins varied in expression and bystander uptake by uninfected cells but could provide insight into bystander toxicity. Conclusions: The results found were present irrespective of cART regimen and systemic viral replication but suggested that these viral reservoirs are a major barrier to curing HIV and treating associated neurocognitive disorders.