As HIV-1 infection has transformed from a fatal crisis into a chronic, life-long disease managed by anti-retroviral therapy (ART), attention has shifted towards curative therapies. A regimen which may allow for viral control off ART will likely require a combination of agents to address HIV reservoirs and induce immune control. These complex interactions are difficult to study in vitro and thus require access to in vivo models. Infection of Rhesus macaques with SIV or chimeric HIV allows study of viral pathogenesis, biomarkers, and emerging cure interventions. Here we describe efforts to apply in situ hybridization (ISH) and immunohistochemistry (IHC) imaging techniques, and ultrasensitive immunoassays (Simoa) to detect host and viral proteins and RNA in samples derived from infected non-human primates (NHPs).
We collected biological samples from lymph nodes (LNs), gut associated lymphoid tissue (GALT), and blood from a cohort of NHPs including uninfected and SHIV162p3-infected viremic and aviremic animals. We measured plasma viral loads by quantitative PCR (assay LOD<10^2 c/ml) and used Simoa to detect SIV-p27 in blood and tissues. Other tissue specimens were fixed, paraffin embedded, and subjected to microtomy for the detection of SIV-p27, viral RNA and follicular dendritic cells (FDCs) by ISH or IHC with commercially available reagents followed by computational image analysis.
We detected SIV-RNA signal by ISH in LNs and GALT of animals with low to undetectable plasma viremia above the background of tissue from uninfected individuals. SIV-p27 IHC signal was detected in LNs of some animals with high viremia, but less efficiently in other tissue compartments and aviremic animals. LNs from animals with plasma viremia >10^3 c/ml presented co-compartmentalization of SIV-RNA and-p27 with FDCs. SIV-p27 measured by Simoa was not readily detected in blood cells but could be detected in rectal and jejunal biopsies of animals with higher viral loads.
Tissue SIV-RNA and p27 ISH/IHC markers quantification correlated in samples across the compartments tested. Using ISH/IHC, Simoa, and qPCR assays, we are assembling a set of biomarkers to investigate viral RNA and protein in NHPs with variable levels of viremia. These assays will also be multiplexed with host biomarkers to further characterize the microenvironments that sustain virus persistence and provide insights into the effectiveness of clearance of infected cells from tissues.