Abstract
The establishment of latent infection and poorly characterized viral reservoirs in tissues represent major obstacles to a definitive cure for HIV. Non-human primate (NHP) models of HIV infection are critical to elucidate pathogenic processes and an essential tool to test novel therapeutic strategies. Thus, the availability of novel assays to measure residual viral replication and reservoirs in NHP models may increase their utility in the search for an HIV cure. We developed a tat/rev induced limiting dilution assay to measure the frequency of CD4+ T cells that express multiply-spliced(ms)_SIV RNA in presence and absence of stimulation. We validated the assay using cell lines and cells from blood and lymph nodes of SIV infected macaques. In vitro, SIV/SHIV TILDA detects only cells expressing viral proteins. In SIV/SHIV-infected macaques, CD4+ T cells that express msSIV/SHIV RNA (TILDA data) were detected also in the setting of very low/undetectable viremia. TILDA data were significantly higher after stimulation and correlated with plasma viral load (pVL). Interestingly, TILDA data from early cART initiation correlated with peak and AUC pVL post-cART interruption. In summary, we developed an assay that may be useful in characterizing viral reservoirs and determining the effect of HIV interventions in NHP models.
Highlights
Despite the advances in understanding the viral reservoir in blood, viral reservoirs in tissues remain poorly characterized
We focused on developing and validating an SIV/SHIV tat/rev induced limiting dilution assay (TILDA) assay that could be used with the SIVmac251/mac[239] and SHIVAD8OE viruses, since they are widely employed in Non-human primate (NHP) studies focused on evaluating novel therapeutic strategies for HIV
Cells are distributed in 22 replicate wells and a nested-PCR amplifying tat/rev transcripts is directly performed without RNA extraction as in the HIV-TILDA
Summary
Despite the advances in understanding the viral reservoir in blood, viral reservoirs in tissues remain poorly characterized. A variety of techniques are currently employed to characterize the viral reservoir in HIV infected humans[19] They include assays that directly measure cell-associated (CA)-viral DNA or RNA with more recent assays including the ability to distinguish ‘intact proviruses’ with the theoretical capability to replicate, from defective proviruses[20]. Among these techniques there is the tat/rev induced limiting dilution assay (TILDA), which measures the frequency of CD4+ T cells producing viral multiply-spliced RNA (msRNA) transcripts either in absence of stimulation or following stimulation with a potent mitogens (phorbol 12-myristate 13-acetate -PMA - and ionomycin) using serial dilutions of input CD4+ T cells[26] This strategy gives estimates of viral reservoir that are between the underestimates of the QVOA and the overestimates of the cell associated viral DNA and unspliced (us)RNA19. The SIV/SHIV TILDA is an informative additional tool in the investigation of viral reservoirs in NHP models to complement other novel, sensitive strategies such as the p27 Simoa[25] and in situ hybridization approaches[30]
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