Intact HIV proviruses persist in the brain despite viral suppression with antiretroviral - PowerPoint Presentation

1. Intact HIV proviruses persist in the brain despite viral suppression with antiretroviral therapyOAA03 ‘Finding the needle in the haystack: Progress in understanding the HIV reservoir’Michael Roche, PhD Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infections and Immunity, AustraliaRMIT University, School of Health and Biomedical Sciences, Australia

2. Conflict of interest disclosureI have no relevant financial relationships with ineligible companies to disclose.

3. The frequency of HIV persistence in the CNS following ART is currently unclearHIV and the brainWhile the advent of ART has reduced the incidence of most severe forms of HIV-associated neurocognitive disorders (HAND) (i.e. encephalitis and dementia), milder forms of HAND persist in 30-69% of people with HIV on ART2-4Cells of the CNS (i.e. microglia5, astrocytes6, neurons etc) are long lived  potential long-term reservoir of HIV in the CNS7,8 Poor penetrance of current ART drugs9,10 and possible immune privileged site.1. Achim et al, J Neuropathol Exp Neurol 1994; 2. Heaton et al, Neurology 2010; 3. Gott et al, Plos One 2017; 4. Simioni et al, AIDS 2010; 5. Reu et al, Cell Rep 2017; 6. Sofroniew Acta Neuropathol 2010; 7. Wallet et al, Front Cell Infect Microbiol. 2019; 8. Sreeram et al, TREIMM 2022; 9. Schnell et al, PLoS Pathog. 2010; 10. Strazielle et al, Rev Med Virol. 2005

4. vRNA and vDNA has been detected in the CNS of NHPs Viral nucleic acid1 and replication competent virus2 is not reduced by ART vRNA and vDNA has been detected in human brain tissue 1,3Localized to microglia4 and astrocytes5The CNS is a potential reservoir of HIV on ART in humans and non-human primates(Byrnes et al. poster PESUA24)The replication competent nature of HIV in the CNS remains unclear(Estes et al, Nature Med. 2017)1. Estes et al, Nat Med 2017; 2. Avalos et al, Mbio 2017; 3. Lamers et al, J Virol 2016; 4. Ko et al, JNIP 2018; 5. Lutgen et al, Plos Pathogens 2020

5. Aim: To characterise the intact and defective CNS reservoir in people living with HIV on ART.We hypothesise that the CNS of people with HIV on ART will possess intact genomes with the potential to be reactivated to produce infectious HIV. Aims and Approach

6. ApproachUsing droplet digital PCR, IHC and LCM with post-mortem CNS and matched lymphoid tissue (NNTC, USA), determine:Size of HIV CNS reservoir Size of intact and defective HIV reservoirCharacterise cellular reservoirs of HIV in the CNSNeg: n=6PWH ART: n=12PWH no ART: n=18

7. Tissue cohortCNS tissue and matched lymphoid tissues Fresh frozen/FFPE autopsy frontal lobe (deep white matter) Donors were suppressed for >2 years on ART with undetectable plasma/CSF viral loadCochrane Ann Neurol 2022, in pressNegNo ARTARTPWH

8. HIV persists in the CNS of PWH despite viral suppression with ARTMann-Whitney U test; P<0.05 statistically significantViral suppression in blood does not affect the level HIV DNA in the CNSCochrane Ann Neurol 2022, in pressQuantification of HIV pol DNA using ddPCRCorrelation of HIV pol DNA in CNS vs HIV plasma viral loadNo ARTARTPWHNeg

9. Does the CNS harbour intact HIV proviruses?CNS:HIV RNA has been detected in CSF of PWH on ART2Brain myeloid cells isolated from NHPs harbour replication competent proviruses by qVOA31. Bruner et al, Nature 2017; 2. Winston et al, AIDS 2019; 3. Avalos et al, MBio 2017Blood:~93% of HIV proviruses are ‘defective’ (unable to produce infectious virions1) in the blood reservoirWhether the CNS of PWH contains intact proviruses is unclear

10. Intact Proviral DNA Assay (IPDA)Multiplex droplet digital PCR method for quantifying intact and defective proviruses1. Probes identify >90% of deleted proviruses as defective:packaging signal (Ψ)EnvIPDA correlates with quantitative viral outgrowth assays1,2,31. Bruner et al, 2019; 2. Faclinelli et al 2020; 3. Papasavvas et al, 2020 Ψ probeenv probeTo define the CNS reservoir the IPDA was utilised

11. The CNS of PWH on ART harbours intact HIV provirusesIPDA results for 10 viremic and 9 virally suppressed PWHCochrane Ann Neurol 2022, in pressIntact HIV proviruses are present at similar levels in the CNS of PWH on and off ARTThe level of intact proviral genomes from matched lymphoid tissues were similar to previous reports1,21. Simonetti et al, PNAS 2020; Martin et al, J Infect Dis 2021)No ARTARTNegNegNo ARTART

12. T cells are unlikely major contributors to HIV in the brainDo T cells in the CNS contribute to the CNS reservoir? T cells in the CNS are not the major contributor of the CNS HIV reservoirNon parametric Spearman correlation; P<0.05 statistically significantCochrane Ann Neurol 2022, in pressCD3+ T cell (#)T cell receptor (TCR) RNA

13. Myeloid cells in the brain harbour HIV DNAHIV vDNA (DNAscope; red) Iba1 (myeloid cell marker; green)Cochrane Ann Neurol 2022, in pressCNS myeloid cells from PWH on ART harbour HIV DNA

14. Myeloid cells in the CNS are a cellular reservoir of HIV in the brainCochrane Ann Neurol 2022, in pressCD68+ myeloid cells isolated from PWH +/- ART (n=7)Nested PCR for HIV V3 and phylogenetic analysis performedSequences were isolated from the CNS compartments of the same PWHHIV V3 sequences in purified myeloid cells from untreated and ART suppressed (n=5)To confirm the presence of HIV DNA in myeloid cells, we performed laser capture microdissection to purify myeloid cell nucleiMyeloid cells in the CNS harbour unique HIV sequences

15. HIV pol DNA is present in multiple brain regions in PWH on ARTddPCR for HIV pol DNAHIV pol DNA was detected in several brain regionsDetection of intact proviral genomes in other brain regions is ongoingMultiple brain regions contain a potential CNS reservoir

16. This study provides evidence of an intact proviral HIV reservoir in the CNS despite long-term viral suppression, and highlights the importance of considering the CNS in all HIV cure strategies In summary…HIV DNA persists in the CNS despite ART Effects cellular activation and HAND?May reseed the periphery?Intact proviral genomes are present in the CNS in people living with HIV following >6.2 years viral suppression. CNS resident myeloid cells harbour HIV DNARole of T cells unclear, unlikely to be major contributors to HIV viral DNA in the brainOther CNS cell types as a HIV reservoir?Considerations:Was ART continued up to death?Is proviral DNA is transcriptionally active?

17. AcknowledgementsNeuroinfections GroupProf Melissa ChurchillDr Thomas AngelovichDr Catherine CochraneSarah ByrnesEmily Waring Jingling ZhouJanna Jamal EddineOregon Health and Science UniversityProf Jacob EstesWe are thankful for the donors to the NNTCPeter Doherty InstituteDr Matthew GartnerProf Sharon LewinCarolin TumpachSt Vincent’s HospitalProf Bruce Brew1R21DA055489-01