Research ArticleAIDS/HIV Open Access | 10.1172/JCI176358

Laura P. Kincer,1 Ameet Dravid,2,3 Mattia Trunfio,4 Andrea Calcagno,4,5 Shuntai Zhou,1 Riccardo Vercesi,6 Serena Spudich,7 Magnus Gisslen,8,9,10 Richard W. Price,11 Paola Cinque,6 and Sarah B. Joseph1,12,13

1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

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1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

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1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

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1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

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1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

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1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

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1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

Find articles by Spudich, S. in: JCI | PubMed | Google Scholar |

1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

Find articles by Gisslen, M. in: JCI | PubMed | Google Scholar |

1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

Find articles by Price, R. in: JCI | PubMed | Google Scholar

1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

Find articles by Cinque, P. in: JCI | PubMed | Google Scholar

1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

2Department of Medicine, Poona Hospital and Research Center, Pune, India.

3Ruby Hall Clinic, Pune, India.

4Unit of Infectious Diseases, Department of Medical Sciences, University of Turin at the “Amedeo di Savoia” Hospital, Torino, Italy.

5ASL “CIttà di Torino,” Torino, Italy.

6Unit of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

7Department of Neurology, Yale University, New Haven, Connecticut, USA.

8Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.

9Department of Infectious Diseases, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

10Public Health Agency of Sweden, Solna, Sweden.

11Department of Neurology, University of California San Francisco, San Francisco, California, USA.

12Department of Microbiology and Immunology and

13UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Sarah B. Joseph, University of North Carolina at Chapel Hill, 22-065 Lineberger Cancer Center, Chapel Hill, North Carolina 27599-7295, USA. Phone: 919.966.5757; Email: sbjoseph@email.unc.edu.

Authorship note: PC and SBJ contributed equally to this work.

Find articles by Joseph, S. in: JCI | PubMed | Google Scholar |

Authorship note: PC and SBJ contributed equally to this work.

Published October 1, 2024 - More info

Published in Volume 134, Issue 19 on October 1, 2024
J Clin Invest. 2024;134(19):e176358. https://doi.org/10.1172/JCI176358.
© 2024 Kincer et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 1, 2024 - Version history
Received: December 11, 2023; Accepted: August 6, 2024 View PDF Abstract

During antiretroviral therapy (ART), most people living with HIV-1 have undetectable HIV-1 RNA in their plasma. However, they occasionally present with new or progressive neurologic deficits and detectable HIV-1 RNA in the cerebrospinal fluid (CSF), a condition defined as neurosymptomatic HIV-1 CSF escape (NSE). We explored the source of neuropathogenesis and HIV-1 RNA in the CSF during NSE by characterizing HIV-1 populations and inflammatory biomarkers in CSF from 25 individuals with NSE. HIV-1 populations in the CSF were uniformly drug resistant and adapted to replication in CD4+ T cells, but differed greatly in genetic diversity, with some having low levels of diversity similar to those observed during untreated primary infection and others having high levels like those during untreated chronic infection. Higher diversity in the CSF during NSE was associated with greater CNS inflammation. Finally, optimization of ART regimen was associated with viral suppression and improvement of neurologic symptoms. These results are consistent with CNS inflammation and neurologic injury during NSE being driven by replication of partially drug-resistant virus in CNS CD4+ T cells. This is unlike nonsuppressible viremia in the plasma during ART, which typically lacks clinical consequences and is generated by virus expression without replication.

Graphical Abstract Introduction

The CNS is an immune-privileged compartment where inflammation is typically limited due to its potential pathogenic effects. For example, neuroinflammation is known to be a contributor in the development of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis (1). In addition, neuroinflammation in response to viral, bacterial, or fungal pathogens can generate severe neuropathogenesis and encephalitis (2).

During untreated infection, HIV-1 replication in CD4+ T lymphocytes and myeloid cells (macrophages and microglia) in the CNS is associated with inflammation (3) and severe neurologic and neurocognitive complications (4, 5). Myeloid lineage cells are prevalent in the CNS, and HIV-1 infection of myeloid cells in the CNS is a central feature of HIV encephalitis in late-stage untreated infection manifesting as HIV-associated dementia (HAD) (4, 5). In contrast, CD4+ T cells are typically rare in the brain (6, 7), though it is now appreciated that they are always present in the CNS (6), including at elevated levels during early HIV-1 infection (8, 9). This often corresponds with a rise in CD8+ T cells within the CNS (10–13).

The implementation of antiretroviral therapy (ART) has greatly reduced rates of severe neurocognitive impairment (14–16) and typically suppresses viral RNA to undetectable levels in both the periphery and CNS (17). Despite these improvements, mild neurocognitive impairment and depression remain common in people living with HIV (PLWH) (14–16), and HIV-1 RNA can occasionally be detected in cerebrospinal fluid (CSF) (18, 19) or brain tissue (20, 21) collected from people on ART. There are many mechanisms that may explain persistent neuropathogenesis during ART, including damage that took place prior to ART, continued virus replication and neuroinflammation, toxicity of ART, coinfections, lifestyle causes, or other underlying factors (reviewed in ref. 22). Studies have, however, observed that neuropathogenesis during ART is correlated with the presence of HIV-1 in the CNS (23). For example, detection of HIV-infected cells in the CSF is associated with poorer performance on neuropsychological tests (24), and HIV-1 cell–associated RNA transcripts in CSF cells are associated with brain injury (19). Therefore, there is a great interest in understanding the source of virus in the CNS during ART and whether it contributes to neuropathogenesis.

Residual viral RNA in the CNS during ART can either be due to ongoing virus replication or virus expression without replication. Replication can occur if the virus is drug resistant or if drug levels in the CNS are noninhibitory. However, ART intensification studies suggest that the latter is not a major driver of detectable levels of CSF HIV-1 RNA in most people on ART (25, 26). Alternatively, virus in the CSF could be generated by infected cells expressing HIV-1 virions in the absence of replication. Such virions could be infectious but unable to infect other cells because ART prevents entry, reverse transcription, nuclear import, and/or integration. Alternatively, virions could be noninfectious due to virion defects, including being immature if produced in the presence of protease inhibitor (PI). The possibility that virions in the CSF could be produced without replication is consistent with studies showing that HIV-1 RNA in the blood during ART is often defective (27, 28) and produced by expression, not replication (27–33).

A rare subset of PLWH and people on ART experience CSF escape in which HIV-1 RNA levels are suppressed in the blood, but remain elevated in the CSF. CSF escape can occur in the absence of neurologic symptoms (i.e., asymptomatic CSF escape [ASE], refs. 34, 35) or with severe neurologic symptoms (i.e., neurosymptomatic HIV-1 CSF escape [NSE], ref. 10–13). We previously performed a detailed genetic and phenotypic analysis of 2 individuals with ASE (18) and observed that one participant had episodic escape in which the CSF contained a homogeneous virus population likely produced by an expanding CD4+ T cell clone in the absence of replication. In contrast, the second participant had a persistent, drug-resistant population of macrophage-tropic virus in the CSF produced by sustained replication in macrophage/microglia during ART (18). These results suggest that ASE may emerge via multiple mechanisms.

This study was designed to identify the cellular source of NSE virus, the conditions needed for NSE, and potential mechanisms generating severe neurologic symptoms in these individuals. We addressed these questions in a rare cohort of 25 PLWH on ART who all met our definition of NSE, i.e., manifested new or worsening neurologic symptoms and signs despite well-controlled blood plasma HIV-1 RNA (i.e, less than 500 HIV-1 RNA copies/ml) and CSF HIV-1 RNA concentrations more than 0.5 logs greater than HIV-1 RNA concentrations in the plasma. We analyzed samples collected at the time of NSE diagnosis for all participants, and additional NSE time points were analyzed for a subset of participants. We used sequencing and phylogenetic analyses to assess viral diversity and determine whether NSE virus is produced by HIV-1 replication or continued virus production from CNS cells without replication and whether NSE virus was drug resistant. We also examined the cellular tropism of NSE virus in order to infer the cell type likely producing NSE virus. Finally, we used CSF biomarkers to assess the inflammatory environment during NSE and explore potential mechanisms that may generate inflammation and severe neurologic symptoms during NSE.

Results

NSE is observed in a subset of PLWH on otherwise suppressive ART. We analyzed CSF from 25 study participants (Table 1) living with HIV-1 on ART who presented with a range of neurological symptoms and complaints (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI176358DS1). All 25 participants were diagnosed clinically with NSE using the following criteria: (a) clinical presentation with new neurologic symptoms and signs without an alternative cause, (b) taking ART with suppression of plasma HIV-1 RNA to below 500 HIV-1 RNA copies/ml, and (c) a CSF HIV-1 RNA greater than that of plasma HIV-1 RNA. Archived CSF samples were collected between 2000 and 2019 at 5 sites: San Francisco, California, USA (n = 7), Pune, India (n = 12), Milan and Turin, Italy (n = 3 and n = 1, respectively), and Gothenburg, Sweden (n = 2).

The main clinical features of the NSE cohort are shown in Table 1. ART regimens and drug-resistance mutations are shown in Table 2. The median CSF HIV-1 RNA was 1,700 copies/ml, and the median plasma HIV-1 RNA was 86 copies/ml. Most (92%, 23/25) participants were on a PI-based ART regimen. Atazanavir (ATV) was very common among those on a PI-containing regimen (74%, 17/23). The median and nadir blood CD4+ T cell counts were 427 cells/μL and 98 cells/μL, respectively. Additionally, all participants examined (24/24) had elevated CSF white blood cells (5 cells or more per μL), with a median of 14 cells/μL (Table 1).

Table 2

ART, drug resistance, and entry phenotypes for NSE participants

To better understand the source of NSE virus and symptoms, we compared clinical and virologic characteristics of participants with NSE to characteristics of participants in 4 cohorts of PLWH (Supplemental Table 2). (a) The first cohort consisted of those with untreated primary HIV-1 infection (n = 7). Participants were from a previously described cohort of people enrolled in the US within 12 months of infection and ART naive (5). (b) The second cohort consisted of those with untreated chronic HIV-1 infection (n = 7). Participants were enrolled in the Tropism of HIV-1 Inflammation and NeuroCognition (THINC) study. All participants had CD4+ T cell counts below 400 cells/μl, were ART naive, and lacked neurologic symptoms. (c) The third cohort consisted of those who were ART-treated and virologically suppressed (n = 49). All participants had HIV-1 RNA levels in the blood and CSF that were undetectable by standard clinical assays and lacked neurologic symptoms. (d) The fourth cohort consisted of 2 previously characterized people with ASE (18). Both individuals were on ART and neurologically asymptomatic with low levels of HIV-1 RNA in plasma and elevated levels in CSF (18).

HIV-1 genetic diversity in the CSF varies greatly across participants with NSE and can reach levels observed in the blood during chronic infection. Single genome amplification (SGA) (36) and/or Illumina MiSeq Deep Sequencing with Primer ID (37) was used to genetically characterize HIV-1 RNA in the CSF of all 25 participants with NSE and in 4 comparator cohorts (cohorts described in Supplemental Table 2). SGA analyses yielded full-length Env glycoprotein (env) sequences, and MiSeq Deep Sequencing with Primer ID generated partial env sequences (521 bases in length) and sequences for 3 additional amplicons used to assess drug resistance (described below). We and others have previously used these approaches to assess genetic diversity in HIV-1 populations (18, 38, 39) and compartmentalization in the CSF (5, 9, 40, 41).

One of the primary goals of this study was to estimate the amount of diversity that accumulated in the CNS during NSE. We first calculated pairwise distance (PWD) across all lineages (Figure 1A). If NSE populations were established by multiple, genetically distinct viruses, then PWD values would represent both diversity contributed by the founders and diversity that accumulated during NSE. To minimize the contribution from founders, we identified individuals with multiple peaks, including a peak with high PWD and a phylogenetic tree with distinct major lineages (Figure 2). This is the pattern expected if an NSE population is established by multiple founder viruses. For these individuals, we recalculated PWD values within each major lineage (Figure 1A) and used the mean of those values (Figure 1A) for all subsequent analyses (when multiple major lineages were present, downstream analyses were performed using the mean of within–major lineage PWD values). While we can’t know for sure if an NSE population was founded by multiple variants, this approach reduces the impact of multiple founder viruses on PWD and generates conservative PWD estimates that better represent diversity accumulated in the CNS during NSE.

Figure 1

HIV-1 NSE populations can be very genetically diverse. (A) To facilitate plotting on a log scale, a constant (0.005) was added to all PWD values and then log transformed. PWD was calculated across all lineages in the CSF of NSE participants (blue) and untreated controls (gray). When multiple major lineages were present in a CSF NSE population, PWD was also calculated within each major lineage, thus avoiding between-lineage comparisons (orange). Means are marked with a vertical line in orange or blue. For individuals with both types of PWD calculations (shown in blue and orange), the mean value of the within-lineage comparison (orange) was used for downstream analyses. A PWD value of 0.004 roughly separated the diversity observed during primary infection from levels observed during chronic infection. The value corresponding to this cutoff is marked with a vertical gray line. Participants with mean PWD above 0.004 are marked with an asterisk. (B) Mean PWD was calculated for the CSF of 2 participants with ASE (PWD less than 0.004, purple closed diamond; PWD above 0.004, purple open diamond; ref. 18) and 25 participants with NSE (PWD below 0.004, gray closed diamonds; PWD above 0.004, black open diamonds). Median is shown with the horizontal bar. (C) Mean PWDs of lower (n = 14) and higher (n = 11) diversity NSE populations were compared with blood- (closed red triangles, n = 7) and CSF-derived (closed blue circles, n = 7) virus from untreated primary infection and blood- (open red triangles, n = 7) and CSF-derived (open blue circles, n = 7) virus from untreated chronic infection. **P < 0.001; ***P < 0.0001; ****P < 0.0001, Mann-Whitney U test. Median value of the mean PWDs is shown with the horizontal bar.

Figure 2

Phylogenetic trees of HIV-1 NSE populations. Genetic diversity during HIV-1 NSE was assessed using SGA and/or Illumina MiSeq Deep Sequencing with Primer ID. CSF-derived (in blue) partial env neighbor-joining phylogenetic trees of participants with NSE. Trees are ordered (in rows) from lowest to highest mean PWD. Asterisks designate the “higher diversity” participants with mean PWD above 0.004.

When we analyzed samples collected from untreated people (Supplemental Table 2), we observed that a mean PWD of 0.004 roughly distinguished levels of diversity observed in primary infection from those observed during chronic infection (Figure 1A). All samples collected during untreated chronic infection had a mean PWD greater than 0.004, and most samples collected during untreated primary infection (the first year after transmission) had a mean PWD below 0.004 (Figure 1A). Therefore, we used a PWD of 0.004 to separate NSE participants into lower and higher diversity groups. The lower diversity group (PWD below 0.004) had levels of diversity similar to those observed during primary untreated infection and in an individual with T cell–tropic ASE (Figure 1, B and C). In contrast, the higher diversity group (PWD above 0.004) had diversity levels similar to those observed during chronic untreated infection and in an individual with persistent, macrophage-tropic ASE (Figure 1, B and C). This somewhat arbitrary division allowed us to compare diversity during NSE to diversity observed at different stages of disease (Figure 1C) and allowed us to determine whether the inflammatory environment was related to the amount of genetic diversity in the NSE population (described below).

HIV-1 RNA levels in the CSF are associated with more diverse NSE populations. PWD levels were not significantly associated with plasma HIV-1 RNA, CSF:blood HIV-1 RNA log10 Δ, CSF WBC count, or blood CD4+ T cell count at the time of escape, but were associated with higher HIV-1 RNA levels in the CSF (Figure 3). Similarly, when NSE participants were grouped by diversity, the median of the mean PWD estimates in the higher diversity group was significantly greater than that of the lower diversity group (Supplemental Figure 1; Mann-Whitney U test, P < 0.0001).

Figure 3

Mean PWD is correlated with CSF HIV-1 RNA. Linear regressions were performed to look for relationships between mean PWD and plasma HIV-1 RNA (A), CSF HIV-1 RNA (B), CSF: plasma HIV-1 RNA log10 Δ (C), blood CD4+ T cell count (D), nadir blood CD4+ T cell count (E), and CSF WBC count (F). The relationship between mean PWD and CSF HIV-1 RNA (B, R2 = 0.3536, P = 0.0089) was the only statistically significant comparison. All P values were adjusted for multiple comparisons.

A subset of participants (23 of 25, Supplemental Table 1) in the NSE cohort were tested for CNS coinfections as part of clinical care and/or a research study (42). Analysis of CSF samples revealed that 9 of 23 people were positive for 1 or more agents, with 8 having detectable EBV in the CSF. Since EBV was the primary coinfecting agent identified in this cohort, we determined whether EBV was related to HIV-1 levels and/or inflammation in the CNS during NSE. We divided the 18 people tested for EBV into those who were positive (n = 8) and those negative (n = 11) for EBV. EBV coinfection was not significantly associated with HIV-1 RNA in the plasma or CSF, CSF:blood HIV-1 RNA log10 Δ, mean PWD, blood CD4+ T cell count at the time of escape, nadir CD4+ T cell count, and/or CSF WBC count (Supplemental Figure 2).

Virus in the CSF during NSE is typically resistant to some, but not all, drugs in the ART regimen. In this study, drug resistance was assessed based on partial integrase, protease, and reverse-transcriptase sequences generated by Illumina MiSeq Deep Sequencing with Primer ID (37). A total of 91% (21/23) of participants analyzed were at least partially resistant to their current ART regimen, but only 15% (4/23) had a resistance mutation to all drugs in their regimen (Table 2). Further, of the 5 participants in this cohort with longitudinal sampling (Table 3), 1 participant (no. 351) was observed to lose and acquire drug-resistance mutations during the study. The complex changes in drug resistance in this participant are consistent with viral replication in the CNS in the presence of low drug levels and a period of treatment interruption (See Table 3).

Table 3

NSE participants with multiple time points

NSE typically resolves if ART is changed to a regimen to which the CSF viral population is sensitive (i.e., ART optimization). ART optimization was typically associated with reductions in HIV-1 RNA in the CSF and improved neurologic symptoms (Supplemental Table 3). In contrast, when ART was not optimized, drug-resistant NSE virus persisted in the CNS (Table 3 and Supplemental Figure 3). Given that ART stops viral replication, but not viral production, these results suggest that during NSE, HIV-1 RNA in the CSF is being produced by replication that contributes to neurologic symptoms. One limitation of this interpretation is that not all follow-up visits took place soon after ART optimization.

NSE virus is adapted to replication in CD4+ T cells. HIV-1 replication in the CNS can take place in resident or trafficking CD4+ T cells (4, 9, 18, 43) or in resident CD4+ macrophage or microglia (4, 18). Using Affinofile cells (44) and our established protocol (45), we assessed the ability of NSE virus to enter cells with low levels of CD4 on the surface. We tested 32 patient-derived envs from 17 NSE participants with different levels of diversity in the NSE population and all were found to require a high density of CD4 for entry (Figure 4A), similar to the levels on CD4+ T cells (46), and were unable to efficiently infect cells expressing low CD4 levels like those found on macrophage/microglia. Thus, we conclude that the virus detected in the CSF during NSE is adapted to replication in CD4+ T cells. Further, the frequency of WBCs in the CSF was elevated during NSE relative to levels found in people on suppressive ART (Mann-Whitney U test, P < 0.001; Figure 4B).