HIV JR-CSF Challenge in Humanized Mice

Stocks of HIV JR-CSF were prepared as previously described [50 (link)–55 (link)] and standardized by p24 ELISA. Humanized BLT mice were challenged i.v. with HIV JR-CSF (100 ng of p24 or 10⁴ Median Tissue Culture Infectious Doses (TCID₅₀). We used a “simple randomization” for the 4 arms by choosing randomly 8 mice per arm. Three weeks post-HIV challenge, infection was confirmed by quantifying viral RNA by PCR viral load in peripheral blood (plasma) using one-step reverse transcriptase quantitative real-time PCR (qRT-PCR) (ABI custom TaqMan Assays-by-Design) according to the manufacturer’s instructions. Primers were 5-CATGTTTTCAGCATTATCAGAAGGA-3 and 5-TGCTTGATGTCCCCCCACT-3, and MGB-probe 5-FAM-CCACCCCACAAGATTTAAACACCATGCTAA-Q-3, where FAM is 6-carboxyfluorescein as we recently described [51 (link)–52 (link)]. The assay sensitivity was of 423 RNA copies per mL of plasma. For quantification of HIV RNA loads in tissues, RNA was extracted from at least 2x 10⁶ CD4+ cells isolated from the harvested tissues using EasySep^™ Human CD4+ T Cell Isolation Kit (STEMCELL Technologies) and the RNeasy Mini Kit (Quiagen) and viral loads quantified by qRT-PCR as described above.