Probes for HIV

Human Immunodeficiency Virus (HIV) persistence in blood and tissue reservoirs including the brain is a major barrier to HIV cure and possible cause of comorbid disease. However, the size and replication competent nature of the CNS reservoir is unclear. Here we used the intact proviral DNA assay (IPDA) to provide the first quantitative assessment of the intact and defective HIV reservoir in the brain of people with HIV (PWH).Total, intact and defective HIV proviruses were measured in autopsy frontal lobe tissue from viremic (n=18) or virologically suppressed (n=12) PWH. Total or intact/defective proviruses were measured by detection of HIV pol or the IPDA, respectively, through use of droplet digital PCR (ddPCR). HIV-seronegative individuals were included as controls (n=6).Total HIV DNA was present at similar levels in brain tissues from untreated viremic and antiretroviral (ART)-suppressed individuals (median: 22.3 vs 26.2 HIV pol copies/106 cells), reflecting a stable CNS reservoir of HIV that persists despite therapy. Furthermore, 8/10 viremic and 6/9 virally suppressed PWH also harbored intact proviruses in the CNS (4.63 vs 12.7 intact copies/106 cells). Viral reservoirs in CNS and matched lymphoid tissue were similar in the composition of intact and/or defective proviruses, albeit at lower levels in the brain. Importantly, CNS resident CD68+ myeloid cells in virally suppressed individuals harbored HIV DNA, directly showing the presence of a CNS resident HIV reservoir.Our results demonstrate the first evidence for an intact, potentially replication competent HIV reservoir in the CNS of virally suppressed PWH. This article is protected by

There is a significant overlap between HIV infection and substance use disorders. Dopamine (DA) is the most abundantly upregulated neurotransmitter in Methamphetamine abuse, with receptors (DRD1-5) that are expressed by neurons as well as by a large diversity of cell types, including innate immune cells that are targets of HIV infection, making them responsive to the hyperdopaminergic environment that is characteristic of stimulant drugs. Therefore, the presence of high levels of dopamine may affect the pathogenesis of HIV, particularly in the brain. The stimulation of HIV-latently infected U1 promonocytes with DA significantly increased viral p24 levels in the supernatant at 24 hrs, suggesting effects on activation and replication. Using selective agonists to different DRDs, we found that DRD1 played a major role in activating viral transcription, followed by DRD4, which increased p24 with a slower kinetic. Transcriptome and systems biology analyses led to the identification of a cluster of genes responsive to DA, where S100A8 and S100A9 were most significantly correlated with the early increase of p24 levels following DA stimulation. Conversely, DA increased the expression of these genes’ transcripts at the protein level, respectively MRP8 and MRP14, which form a complex also known as Calprotectin. Interestingly, MRP8/14 was able to stimulate HIV transcription in latent U1 cells, and this occurred via binding to the receptor for advanced glycosylation end-product (RAGE). Using selective agonists, both DRD1 and DRD4 increased MRP8/14 on the surface and in the cytoplasm, as well as secreted in the supernatants. On the other hand, while DRD1/5 did not affect the expression of RAGE, DRD4 stimulation caused its downregulation, offering a mechanistic for the delayed effect via DRD4 on p24 increase. To cross-validate MRP8/14 as a DA signature with biomarker value, we tested its expression in HIV+ Meth users’ brains and peripheral cells. MRP8/14+ cells were more frequently identified in mesolimbic areas such as basal ganglia of HIV+ Meth+ cases compared to HIV+ non-Meth users or to controls. Likewise, MRP8/14+ CD11b+ monocytes were more frequent in HIV+ Meth users, particularly in participants with detectable viral load in the CSF. Overall, our results suggest that the MRP8 and MRP14 complex may serve as a signature to distinguish subjects using addictive substances in the context of HIV, and that this may play a role in aggravating HIV pathology by promoting viral replication in people with HIV who use Meth.

HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals have an increased risk of tuberculosis prior to loss of peripheral CD4 T cells, raising the possibility that HIV co-infection leads to CD4 T cell depletion in lung tissue before it is evident in blood. Here, we use rhesus macaques to study the early effects of simian immunodeficiency virus (SIV) co-infection on pulmonary granulomas. Two weeks after SIV inoculation of Mtb-infected macaques, Mtb-specific CD4 T cells are dramatically depleted from granulomas, before CD4 T cell loss in blood, airways, and lymph nodes, or increases in bacterial loads or radiographic evidence of disease. Spatially, CD4 T cells are preferentially depleted from the granuloma core and cuff relative to B cell-rich regions. Moreover, live imaging of granuloma explants show that intralesional CD4 T cell motility is reduced after SIV co-infection. Thus, granuloma CD4 T cells may be decimated before many co-infected individuals experience the first symptoms of acute HIV infection.

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.