Probes for HIV

HIV can infect multiple cells in the liver including hepatocytes, Kupffer cells and infiltrating T cells, but whether HIV can persist in the liver in people with HIV (PWH) on suppressive antiretroviral therapy (ART) remains unknown.In a prospective longitudinal cohort of PWH and hepatitis B virus (HBV) co-infection living in Bangkok, Thailand, we collected blood and liver biopsies from 18 participants prior to and following ART and quantified HIV and HBV persistence using quantitative (q)PCR and RNA/DNAscope. Antiretroviral (ARV) drug levels were quantified using mass spectroscopy.In liver biopsies taken prior to ART, HIV DNA and HIV RNA were detected by qPCR in 53% (9/17) and 47% (8/17) of participants respectively. Following a median ART duration of 3.4 years, HIV DNA was detected in liver in 61% (11/18) of participants by either qPCR, DNAscope or both, but only at very low and non-quantifiable levels. Using immunohistochemistry, HIV DNA was observed in both hepatocytes and liver infiltrating CD4+ T cells on ART. HIV RNA was not detected in liver biopsies collected on ART, by either qPCR or RNAscope. All ARVs were clearly detected in liver tissue.Persistence of HIV DNA in liver in PWH on ART represents an additional reservoir that warrants further investigation.National Health and Medical Research Council of Australia (Project Grant APP1101836, 1149990, and 1135851); This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024.

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.

HIV-associated nephropathy (HIVAN) impairs functions of both glomeruli and tubules. Attention has been previously focused on the HIVAN glomerulopathy. Tubular injury has drawn increased attention because sodium wasting is common in hospitalized HIV/AIDS patients. We used viral protein R (Vpr)-transgenic mice to investigate the mechanisms whereby Vpr contributes to urinary sodium wasting. In phosphoenolpyruvate carboxykinase promoter-driven Vpr-transgenic mice, in situ hybridization showed that Vpr mRNA was expressed in all nephron segments, including the distal convoluted tubule. Vpr-transgenic mice, compared with wild-type littermates, markedly increased urinary sodium excretion, despite similar plasma renin activity and aldosterone levels. Kidneys from Vpr-transgenic mice also markedly reduced protein abundance of the Na+-Cl- cotransporter (NCC), while mineralocorticoid receptor (MR) protein expression level was unchanged. In African green monkey kidney cells, Vpr abrogated the aldosterone-mediated stimulation of MR transcriptional activity. Gene expression of Slc12a3 (NCC) in Vpr-transgenic mice was significantly lower compared with wild-type mice, assessed by both qRT-PCR and RNAScope in situ hybridization analysis. Chromatin immunoprecipitation assays identified multiple MR response elements (MRE), located from 5 kb upstream of the transcription start site and extending to the third exon of the SLC12A3 gene. Mutation of MRE and SP1 sites in the SLC12A3 promoter region abrogated the transcriptional responses to aldosterone and Vpr, indicating that functional MRE and SP1 are required for the SLC12A3 gene suppression in response to Vpr. Thus, Vpr attenuates MR transcriptional activity and inhibits Slc12a3 transcription in the distal convoluted tubule and contributes to salt wasting in Vpr-transgenic mice.