Baricitinib for Reduction of HIV - CNS

Changes in CSF integrated proviral DNA

The study team will obtain up to 40 milliliters (ml) of CSF from individuals who weigh at least 60 kilograms, otherwise, they will draw up to 30 ml of CSF. CSF integrated proviral DNA (IPDA and Alu-PCR) will be assessed: Integrated proviral DNA will be performed using existing methods in the clinical research group, which employ the Alu-PCR real-time platform, with a limit of 3 copies. Integrated proviral DNA assay (IPDA) to measure the replication-competent reservoir will be used with digital droplet PCR methodology.

Changes in CSF HIV RNA by single copy assay

This method is based on a proprietary protocol which is used in conjunction with a commercial HIV-1 RNA easyQ reagent kit (bioMerieux Inc, Lyon, France). Briefly, a specimen of up to 2 ml of human plasma or CSF is added to lysis buffer containing guanidine thiocyanate. HIV-1 RNA is extracted in combination with the easyMAG platform (bioMerieux, Inc). Eluates containing HIV-1 RNA are aliquoted into 0.5 mL reaction tubes and amplified using 3 enzymes: T7 RNA polymerase, avian myeloblastosis virus reverse transcriptase, and RNase H. Molecular beacons targeting the pol/gag region of HIV-1 RNA are used for amplification and detection by isothermal reactions at 41oC. HIV-1 RNA level is quantified in conjunction with the NucliSENS easyQ HIV-1 v2.0 Director software and a proprietary algorithm developed by bioMONTR Labs. The dynamic range of this HIV-1 assay is 1-5,000,000 copies/mL of 0.73 - 1600 HIV-1 copies /μl. The HIV-1 copy number is normalized per one million of WBCs.

Changes in HIV tat protein in CSF

The study team will obtain up to 40 milliliters (ml) of CSF from individuals who weigh at least 60 kilograms, otherwise we will draw up to 30 ml of CSF. HIV Tat in the CSF will be quantified according to published methods for ELISA to measure HIV Tat in human CSF.

Changes in CSF HIV cell-associated RNA by Double-R assay.

The study team will obtain up to 40 milliliters (ml) of CSF from individuals who weigh at least 60 kilograms, otherwise, they will draw up to 30 ml of CSF. Briefly, after the isolation of Peripheral blood mononuclear cells (PBMC) and cells from CSF, samples will be resuspended in phosphate-buffered saline and stored for shipment in O-ring tubes. RNA will then be extracted using the Maxwell rapid sample concentrator (RSC) automated extraction platform. White Blood Cells (WBC) will be counted after red blood cell lysis using TC20 Automated Cell Counter, and these counts will be used to normalize the HIV-1 RNA results as copy numbers/106 cells. HIV-1 copy number per patient sample, in duplicate, is determined from a standard curve generated with HIV-1 plasmid controls, with a range of 0.73 - 1600 HIV-1 copies /μl. The HIV-1 copy number is normalized per one million of WBCs.

Changes in CSF HIV cell associated CSF HIV DNA by Double-R assay.

The study team will obtain up to 40 milliliters (ml) of CSF from individuals who weigh at least 60 kilograms, otherwise they will draw up to 30 ml of CSF. Briefly, after the isolation of Peripheral blood mononuclear cells (PBMC) and cells from CSF, samples will be resuspended in phosphate-buffered saline and stored for shipment in O-ring tubes. DNA will then be extracted using the Maxwell rapid sample concentrator (RSC) automated extraction platform. WBCs will be counted after red blood cell lysis using TC20 Automated Cell Counter, and these counts will be used to normalize the HIV-1 DNA results as copy numbers/106 cells. HIV-1 copy number per patient sample, in duplicate, is determined from a standard curve generated with HIV-1 plasmid controls, with a range of 0.73 - 1600 HIV-1 copies /μl. The HIV-1 copy number is normalized per one million of WBCs.