Elucidating the Mechanisms Driving the Pathophysiology of HIV-Associated Neurocognitive Disorders

Globally, approximately 40% of people living with human immunodeficiency virus (HIV) on antiretroviral therapy (ART) develop a form of HIV-associated neurocognitive disorder (HAND). Viral persistence and associated neuroinflammation, in addition to ongoing systemic inflammation, are thought to play crucial roles in neuropathology. However, the cellular immune environment in the brain of virally suppressed people with HIV (PWH) is unclear. Here we characterised viral persistence and immune activation in the brain during chronic HIV infection using a simian immunodeficiency virus (SIV) non-human primate (NHP) model and autopsy frontal cortex tissue from PWH. Additionally, matched gut and frontal cortex tissue from SIV+ NHPs and an SIV-uninfected model of chronic gut damage were utilised to determine whether gut damage can impact neuroinflammation alone, in the absence of SIV infection. NHP Cohort: SIV+ NHPs sacrificed during acute (n=4), chronic (n=12) or virally suppressed (VS) SIV infection (n=11) and SIV-uninfected NHPs with (n=4) or without experimentally induced gut damage (n=4). Human Cohort: Human autopsy brain tissue from non-VS (nVS; n=7) or VS PWH (n=10). HIV/SIV viral RNA/DNA and immune activation/inflammation were quantified at a cellular level in matched gut tissue from the SIV cohort and frontal cortex tissue from each cohort using DNA/RNAscope, multiplex immunofluorescence, droplet digital PCR and the intact proviral DNA assay (IPDA), where appropriate. SIV DNA+ and RNA+ cells were detected in the frontal cortex and gut tissue of all SIV+ groups tested. Additionally, the frequency of viral DNA+ cells were not reduced in the frontal cortex or gut in VS NHPs (P< 0.05), supporting the presence of a stable viral reservoir in the frontal cortex and gut tissue that is not reduced by ART. SIV+ NHPs had enhanced type I interferon-induced MX dynamin like GTPase 1 (MX1), oxidative stress and transforming growth factor beta 1 (TGFβ1) responses, expansion of activated astrocytes and myeloid cells as well as reduced blood brain barrier (BBB) integrity compared to uninfected NHPs, which persisted despite ART (P<0.05 for all). Surprisingly, BBB breakdown and neuroinflammation correlated strongly with measures of gut inflammation, but not brain viral load. Similar immune activation profiles were present in the frontal cortex tissue of the SIV uninfected animals with chronic gut damage, indicating that persistent damage to the gut alone can contribute to immune activation in the frontal cortex independent of SIV infection. Comparative analysis in human autopsy frontal cortex tissue from PWH showed similar levels of total HIV DNA and intact proviral DNA in nVS compared to VS PWH (P>0.05 for both), reflecting a stable CNS reservoir of HIV that persists despite viral suppression. Similar to findings in NHPs, frontal cortex tissue from PWH had enhanced Mx1 and tumour necrosis factor alpha (TNFa) responses and expansion of activated myeloid cells that persisted despite viral suppression (P<0.05 for all). In contrast to the limited relationship between local CNS virus and neuroinflammation in NHPs, a strong positive correlation between intact HIV DNA and Mx1+ cells were present in the frontal cortex of VS PWH (p<0.05). While there were no significant correlations present between HIV DNA in the frontal cortex and neurocognitive domain scores, both Mx1 and TGFβ1+ myeloid cells correlated with poorer scores in the attention domain (P<0.05 for both). Our findings in NHPs provide the strongest evidence to date that the frontal cortex of SIV+ NHPs remains in an activated state despite long-term ART treatment and that gut damage can induce neuroimmune activation. In humans, the presence of intact virus rather than total HIV DNA was associated with increased neuroinflammation, which was in turn associated with poorer neurocognitive outcomes, highlighting the ongoing effects of HIV reservoirs in VS PWH. Overall, the data presented demonstrates the frontal cortex as a site of viral persistence and chronic immune activation in PWH and SIV+ NHPs despite long-term viral suppression.