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  Conference on Retroviruses
and Opportunistic Infections
Boston USA
March 8-11, 2020
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  CROI 220
Reported by Jules Levin
Oral Session:
SYSTEM PATHOGENESIS 1 of several talks

Shelli Farhadian1, Joshua C. Cyktor2, Asma Naqvi2, Michael J. Corley3, Jennifer Chiarella1, Rachela Calvi1, Michelle Chintanaphol1, Geoffrey Lyon1, Diane Trotta1, John W. Mellors2, Serena S. Spudich1 1Yale University, New Haven, CT, USA, 2University of Pittsburgh, Pittsburgh, PA, USA, 3University of Hawaii, Honolulu, HI, USA
Background: HIV-infected cells persist in the central nervous system (CNS) in at least half of people with HIV (PWH) on antiretroviral therapy (ART). We previously reported on a novel population of myeloid lineage microglia-like cells in cerebrospinal fluid (CSF) from PWH on ART; however the identity of CNS cells containing proviruses remains unknown.
Methods: Fresh CSF and blood were collected from PWH (median 20yrs on ART, range 4-24yrs). Single cell CITE-seq was performed to validate CD204 as a marker for CSF microglia-like cells. CSF cells and peripheral blood mononuclear cells (PBMCs) were separated using fluorescence activated cell sorting into three subsets based on expression of: CD3+CD4+, CD3+CD8+, and CD3-CD20-CD204+. HIV DNA levels were determined in each subset using a sensitive qPCR assay targeting HIV integrase (iCAD). HIV DNA measurements were normalized for cell equivalents determined by CCR5 qPCR.
Results: Six donors had plasma HIV RNA levels <20 copies/mL; one had 748 copies/mL. Two donors had HIV RNA detected in CSF despite plasma viral suppression, with 95 and 163 copies/mL HIV RNA detected in CSF. The median number of CSF cells obtained per donors was 35,327 (range 13,000-85,000) in 25mL of CSF. HIV DNA was detected in blood CD4+ T cells from 6/7 donors, and not detected in blood CD4+ T cells in one donor. In CSF, HIV DNA was detected in CD4+ T cells in 6/7 donors (of which 5 donors also had HIV DNA detected in blood CD4+ T cells). HIV DNA copies per 1 million cell equivalents was higher (median 7.1 fold, range 0.3-132) in CSF CD4+ T cells than in blood CD4+ T cells in 5/6 donors. No donor had HIV DNA detected in CSF CD8+ T cells.
We isolated genomic DNA from CD204+ CSF cells in three participants and observed that one participant had HIV DNA detected in CD204+ CSF cells. This donor had plasma HIV RNA 748 copies/mL and CSF HIV RNA 87 copies/mL. HIV DNA levels in this participant were 4368 copies per 1 million CD204+ CSF cells, 2769 copies per 1 million in CSF CD4+ T cells, and 401 copies per 1 million blood CD4+ T cells.
Conclusion:We detected HIV DNA in CD4+T and myeloid cells in CSF in a limited sample of PWH on ART. Normalized HIV DNA in CD4+ T cells from CSF was higher than in blood in most donors. Larger studies should assess whether the HIV DNA detected is in replication-competent proviruses, and whether other CNS immune cell types are HIV-infected.