proviral DNA in the CNS of a virally suppressed HIVinfected individual NCHIV meeting Marieke Nühn 22112022 Potential conflict of interests ID: 1042174
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1. Detection of Intact HIV proviral DNA in the CNS of a virally suppressed HIV-infected individualNCHIV meeting Marieke Nühn22-11-2022
2. (Potential) conflict of interestsNoneRelations that could be relevant for the meetingNoneDisclosure of speaker’s interest
3. IntroductionCentral nervous system (CNS) is a potential viral reservoir for HIV1,2Contributing to the development of HIV-associated dementia (HAND)3Foci of infected CNS cells of virally suppressed individuals4,5,61-10% of the microglia/macrophage cells are infectedAre these capable of producing replication-competent virus? Examining genomic intactness: IPDAPhenotypical characterization of viral envelope gene 1. Joseph et al. 2015. 2. Bednar et al. 2015. 3. Rojas-Celis et al. 2019. 4.Churchill et al. 2006. 5.Thompson et al. 2011. 6. Trillo-Pazos et al. 2003.
4. 65-year old maleLong-term ART (undetectable viral load <50cp/mL)At least for 4 yearsDied of pneumonia Post-mortem biopsies of 3 brain regions: Frontal lobe, occipital lobe and the subventricular zonePatient CharacteristicsCreated with: Biorender.com
5. Tissue mincing & mechanical and enzymatic dissociationMicroglia enrichment; CD11b+ isolationMethodsCreated with: Biorender.com
6. Intact Proviral DNA Assay (IPDA)Psi present and 3’ deletion/hypermutationEnv present and 5’ deletionDouble positive intactXX1. Ho et al. 2013. 2. Bruner et al. 2019Most widely-used PCR detects total proviral DNA (intact & defective)1,2 Digital droplet PCR (ddPCR); sample in +/- 20.000 dropletsIPDA: which droplet contains intact proviral DNA?2
7. 13% of proviruses were intactIntact proviral DNA is 2.8 fold higher in microglia-enriched fractionOccipital lobe harbors most intact provirusesDetection of intact HIV DNA within CNS cellsCD11b pos. CD11b neg.FRONTOCCSVZ
8. CD11b+ fraction of frontal lobe; generation of full-length envelope reporter viruses Phenotypical characterization in CD4+ T cellsProductive infection and replication up to 14 daysInhibition by Maraviroc (CCR5 receptor)Replication competent virus?
9. Presence of intact HIV proviral DNA in different regionsIn microglia, but also in non-myeloid cellsCD4+ T cells, astrocytes or residual microglia?Replication competent and require CCR5 receptor for viral entryFuture characterization of HIV in the CNS Persistence mechanismsImpact of HIV-infection on CNS cellsDiscussion & Future research
10. Stephanie GumbsPauline SchipperLavina GharuNinée BuchholtzGijsje SnijdersFrederieke GigaseJori SymonsSusan MorgelloAnnemarie WensingLot de WitteMonique NijhuisAcknowledgementsICD-ICK4HIVCureManhattan HIV Brain Bank