reading assignment 2 HIV topic

RESEARCH ARTICLE

T cell derived HIV-1 is present in the CSF in the

face of suppressive antiretroviral therapy

Gila LustigID 1, Sandile CeleID

2,3, Farina KarimID 2,3, Anne Derache2, Abigail Ngoepe2,

Khadija Khan2,3, Bernadett I. Gosnell4, Mahomed-Yunus S. MoosaID 4, Ntombi Ntshuba2‡,

Suzaan Marais5, Prakash M. Jeena6, Katya GovenderID 2, John Adamson2,

Henrik Kløverpris2,7,8, Ravindra K. GuptaID 2,9, Rohen HarrichandparsadID

10, Vinod

B. Patel5, Alex SigalID 2,3,11*

1 Centre for the AIDS Programme of Research in South Africa, Durban, South Africa, 2 Africa Health

Research Institute, Durban, South Africa, 3 School of Laboratory Medicine and Medical Sciences, University

of KwaZulu-Natal, Durban, South Africa, 4 Department of Infectious Diseases, University of KwaZulu-Natal,

Durban, South Africa, 5 Department of Neurology, University of KwaZulu-Natal, Durban, South Africa,

6 Discipline of Pediatrics and Child Health, University of KwaZulu-Natal, Durban, South Africa, 7 Division of

Infection and Immunity, University College London, London, United Kingdom, 8 Department of Immunology

and Microbiology, University of Copenhagen, Copenhagen, Denmark, 9 Department of Medicine, University

of Cambridge, Cambridge, United Kingdom, 10 Department of Neurosurgery, University of KwaZulu-Natal,

Durban, South Africa, 11 Max Planck Institute for Infection Biology, Berlin, Germany

‡ Unavailable.

* [email protected]

Abstract

HIV cerebrospinal fluid (CSF) escape, where HIV is suppressed in blood but detectable in

CSF, occurs when HIV persists in the CNS despite antiretroviral therapy (ART). To deter-

mine the virus producing cell type and whether lowered CSF ART levels are responsible for

CSF escape, we collected blood and CSF from 156 neurosymptomatic participants from

Durban, South Africa. We observed that 28% of participants with an undetectable HIV blood

viral load showed CSF escape. We detected host cell surface markers on the HIV envelope

to determine the cellular source of HIV in participants on the first line regimen of efavirenz,

emtricitabine, and tenofovir. We confirmed CD26 as a marker which could differentiate

between T cells and macrophages and microglia, and quantified CD26 levels on the virion

surface, comparing the result to virus from in vitro infected T cells or macrophages. The

measured CD26 level was consistent with the presence of T cell produced virus. We found

no significant differences in ART concentrations between CSF escape and fully suppressed

individuals in CSF or blood, and did not observe a clear association with drug resistance

mutations in CSF virus which would allow HIV to replicate. Hence, CSF HIV in the face of

ART may at least partly originate in CD4+ T cell populations.

Author summary

The brain may be a site where HIV persists despite antiretroviral therapy (ART). Persis-

tence can manifest as cerebrospinal fluid (CSF) escape, where HIV is detectable in the

CSF but not the blood in some individuals. The reasons for CSF escape are incompletely

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PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009871 September 23, 2021 1 / 21

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OPEN ACCESS

Citation: Lustig G, Cele S, Karim F, Derache A,

Ngoepe A, Khan K, et al. (2021) T cell derived HIV-

1 is present in the CSF in the face of suppressive

antiretroviral therapy. PLoS Pathog 17(9):

e1009871. https://doi.org/10.1371/journal.

ppat.1009871

Editor: Ronald Swanstrom, University of North

Carolina at Chapel Hill, UNITED STATES

Received: August 13, 2020

Accepted: August 6, 2021

Published: September 23, 2021

Copyright: © 2021 Lustig et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the manuscript and its Supporting

information files.

Funding: This study was supported by National

Institute of Mental Health award R21MH104220

(AS) and National Institute of Allergy and Infectious

Diseases award R01AI138546 (AS). Salary support

was provided to GL and AS from National Institute

of Mental Health award R21MH104220 and

National Institute of Allergy and Infectious Diseases

award R01AI138546. The funders had no role in

understood. Evidence from individuals mostly on second line protease inhibitor-based

ART indicates that detectable HIV in this compartment may have acquired drug resis-

tance. In this work we investigated HIV in blood and CSF of 156 participants from Dur-

ban, South Africa. We observed a very high prevalence of CSF escape of 28%. We aimed

to find the cell type responsible for producing HIV in CSF escape and whether replication

occurred because of lower CSF drug levels or because the virus has developed resistance

to therapy. We found that at least some of the CSF HIV was produced by T cells, and that

drug resistance was not always present. This suggests that at least part of the CSF HIV res-

ervoir may be generated by either an infection mode not requiring drug resistance for

viral replication, or by latently infected CD4+ T cells trafficking to and releasing HIV in

the CSF without extensive viral replication taking place.

Introduction

HIV persistence in the face of ART necessitates lifelong adherence to treatment. The CNS may

serve as one reservoir for HIV persistence [1]. HIV infection in the CNS in the absence of sup-

pressive ART may lead to HIV-associated neurocognitive disorders (HAND). Yet, even in the

presence of ART mediated suppression, sub-clinical cognitive impairment is common [2–7]

and there is widespread immune activation and inflammation in the CNS [8–10]. Consistent

with a role for the CNS as an HIV reservoir, a subset of individuals show CSF escape, where

HIV is detectable in the CSF while being successfully suppressed below the level of detection

in the blood [11–16].

Potential reasons for a CNS reservoir include reduced drug levels. Drug levels of efavirenz

(EFV), emtricitabine (FTC), and tenofovir (TFV) in the CSF are reduced approximately

200-fold, 2-fold, and 20-fold, respectively in the CSF relative to blood [17–19]. Since the

majority of individuals do not show detectable virus in the CSF, these lowered ART levels

seem to be sufficient to suppress viremia. However, it is unclear if ART levels in the CSF are

lower in individuals with neurosymptomatic CSF escape in South Africa, accounting for the

lack of effective suppression in this compartment and possibly evolution of drug resistance

mutations. Mutations could include the M184V or M184I resistance mutation to FTC, a drug

which would provide selective pressure since it has good penetration to the CNS [14, 20–24].

There is evidence for compartmentalized HIV infection in the CNS, indicating that CNS

specific cell subtypes may be involved [25–27]. HIV infected, long lived CNS resident host

cells such as microglia and perivascular macrophages may be responsible for the HIV reservoir

in the CNS [11, 27–31]. HIV infection may not be appreciably cytotoxic in these cells [32] and

these cells are resistant to cytotoxic T lymphocyte killing [33], allowing long-term infected cell

persistence without new cycles of re-infection.

T cells are also present in the CNS. CSF contains trafficking T cells, mostly CD4+ memory

cells, which enter across the choroid plexus [34]. T cell-tropic HIV is present in the CSF in

some individuals [27, 28, 35] and CSF HIV was found to have fast decay kinetics upon ART

initiation, consistent with the short half-lives of infected T cells [36].

Here we aimed to determine the cellular source of HIV in the brain and whether lower

ART levels relative to fully suppressed individuals account for CSF escape in individuals from

Durban, South Africa. This is the first time in our knowledge where CSF escape in Sub-Saha-

ran Africa has been investigated in a relatively large number of participants on ART [37].

Since accessing HIV infected cells from the CNS is challenging, we chose a method which

could determine the cell-of-origin of cell-free HIV sampled from the CSF. Upon viral budding,

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study design, data collection and analysis, decision

to publish, or preparation of the manuscript.

Competing interests: The authors have declared

that no competing interests exist. Author Ntombi

Ntshuba was unable to confirm their authorship

contributions. On their behalf, the corresponding

author has reported their contributions to the best

of their knowledge.

the HIV envelope contains host surface markers [38, 39] since HIV uses the cellular plasma

membrane as its envelope. The host surface markers can be bound with antibodies and

detected using a variety of techniques [40–42] including electron microscopy [39], mass spec-

trometry [43], flow cytometry [44, 45], and immunomagnetic capture [46–50]. Many of these

studies report that HIV derived from macrophage lineage cells expresses CD36 [43–46, 48–

50], a scavenger receptor [51–56], on its envelope. HIV derived from T cells expresses CD26

[44, 46, 48–50], a dipeptidyl-peptidase involved in T cell activation [57]. We tested the ability

of CD26 and CD36 to differentiate between T cells and other cell types. We found that in sam-

ples obtained from the South African study participants, CD26 was T cell specific. CSF escape

HIV had CD26 on its surface, consistent with at least partial T cell origin of CSF escape virus.

We also observed that CSF ART concentrations in CSF escape were not significantly different

from those of participants with viral suppression and did not detect a clear association with

drug resistance mutations, indicating that detectable T cell origin HIV can persist in the CSF

despite suppressive ART.

Materials and methods

Ethical statement

CSF and matched blood were obtained from participants indicated for lumbar puncture

enrolled at Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital in Durban,

South Africa after written informed consent (University of KwaZulu-Natal Institutional

Review Board approval BE385/13). Discarded tissue from the field of neurosurgery was

obtained from two participants enrolled at Inkosi Albert Luthuli Central Hospital indicated

for neurosurgery after written informed consent (University of KwaZulu-Natal Institutional

Review Board approval BE315/18). Blood for PBMC, CD4+ and CD14+ cell isolation was

obtained from adult healthy volunteers after written informed consent (University of Kwa-

Zulu-Natal Institutional Review Board approval BE022/13 and BE083/18). Lymph nodes were

obtained from the field of surgery of participants undergoing surgery for diagnostic purposes

and/or complications of inflammatory lung disease. Informed consent was obtained from each

participant, and the study protocol approved by the University of KwaZulu-Natal Institutional

Review Board (approval BE024/09).

Statistical tests

Data is described with the non-parametric measures of median and interquartile range, and

significance determined using the non-parametric Mann-Whitney U test for pairwise compar-

isons, Fisher exact test for pairwise comparisons of frequencies, and the Kruskal-Wallis test

with multiple comparison correction by the Dunn Method for comparisons involved more

than two populations. All tests were performed using Graphpad Prism 8 software.

CSF sample collection and processing

Fresh CSF and matching blood samples were transported to the laboratory and processed

immediately. Two separate EDTA tubes of 4 mL blood were sent for testing in parallel: one for

a CD4/CD8 count at an accredited diagnostic laboratory (Ampath, Durban, South Africa) and

one for viral load at an accredited diagnostic laboratory (Molecular Diagnostic Services, Dur-

ban, South Africa, using the RealTime HIV-1 viral load test on an Abbott machine). CSF sam-

ples were spun for 10 min at 1000 g to remove debris. CSF supernatant was frozen in 1 mL

aliquots at -80˚C. One aliquot of 100 μl CSF was sent for viral load (Molecular Diagnostic

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Services) directly after the spin. Plasma was processed by spinning the whole blood for 10 min

at 1300 g, no brake. The top layer was removed and stored in 1 mL aliquots at -80˚C.

Antiretrovirals, viruses and cells

The following reagents were obtained through the AIDS Research and Reference Reagent Pro-

gram, National Institute of Allergy and Infectious Diseases, National Institutes of Health: the

antiretrovirals EFV, FTC, and TFV and the plasmid for the macrophage tropic pNL4–3(AD8)

HIV molecular clone. NL4–3 and NL4–3(AD8) HIV stocks were produced by transfection of

HEK293 cells with the molecular clone plasmids using TransIT-LT1 (Mirus) transfection

reagent. Supernatant containing released virus was harvested two days post-transfection and

filtered through a 0.45 micron filter (GVS) and stored in 0.5 mL aliqouts at -80˚C. The number

of HIV RNA genomes in viral stocks was determined using the RealTime HIV-1 viral load test

(Molecular Diagnostic Services, Durban, South Africa). RevCEM-E7 cells were generated as

previously described [58]. Cell culture medium was complete RPMI 1640 supplemented with

L-glutamine, sodium pyruvate, HEPES, non-essential amino acids (Lonza), and 10% heat-

inactivated FBS (Hyclone). PBMCs were isolated by density gradient centrifugation using His-

topaque 1077 (Sigma). CD4+ or CD14+ cells were positively selected using either CD4 or

CD14 Microbeads loaded onto MACS separation columns according to manufacturer’s

instructions (Miltenyi Biotec). CD4+ PBMCs were grown in the above cell media supple-

mented with 5 ng/mL IL-2 (Peprotech) and 10 μg/mL PHA (Sigma-Aldrich). Monocyte-

derived macrophages were grown in RPMI 1640 supplemented with 10% human serum

(Sigma) with added L-glutamine, sodium pyruvate, HEPES, and non-essential amino acids

(Lonza), and differentiated with 20 ng/mL M-CSF (Peprotech) for 10 days.

Surface staining and detection of CD26 and CD36 markers by flow

cytometry

Staining of MDM and PBMC T cells: MDM and CD4+ PBMCs were generated as described

above. PBMCs were washed once in PBS-/-. MDM were washed once in PBS-/- then incubated

in 5 mM EDTA in PBS-/- for 30 minutes on ice. Macrophages were collected by pipetting vig-

orously and the remaining attached cells were removed by gentle scraping. Cells were then

incubated with either CD3-APC and CD8-Bv500 (PBMC) or CD68-APC (MDM) and

CD26-FITC and CD36-PE (Biolegend) fluorescently labelled antibodies in staining buffer

(PBS-/- with 3%FCS) for 30 min on ice. The samples were then washed, resuspended in 400 μL

staining buffer and acquired on a FACSCalibur machine (BD Biosciences). Results were ana-

lyzed using FlowJo software. Staining of LN cells: LN from the field of indicated cardiothoracic

surgery were cellularized by gentle mechanical dissociation and cryopreserved. For staining,

LN cells were thawed, washed once in PBS-/-, then incubated with the following fluorescently

conjugated antibody mix for 30 min: CD45-HV500, CD19-BV785, CD3-PE-CF594,

CD4-AF700 HLA-DR-BV605 (all BD Biosciences), CD26-FITC, CD36-PE (Biolegend), and

the LIVE/DEAD Fixable Near-IR Dead Cell Stain (ThermoFisher Scientific). Cells were then

fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD

Biosciences) according to the manufacturer’s instructions. Cells were then stained with

CD68-APC antibodies (Biolegend). Cells were washed, fixed in 2% formaldehyde and acquired

on a BD ARIA Fusion flow cytometer (BD Biosciences).

Isolation and surface staining of human microglia

Meninges discarded tissue samples were transported immediately to the laboratory for pro-

cessing. The tissue was dissociated (Brain Dissociation kit, Miltenyi). The dissociated cells

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underwent myelin removal (Myelin removal kit, Miltenyi) and CD11b+ cells were further

purified (CD11b microglia Microbeads, Miltenyi). All kits were used according to the manu-

facturers instructions. The cells were then surface stained with fluorescently conjugated anti-

bodies for microglial markers: CD11b-APC, CD45-Bv605 and P2RY12-Bv421, CD26-FITC,

and CD36-PE (Biolegend). The cells were incubated with antibodies for 30 min on ice, washed,

resuspended in 500 μL staining buffer, and acquired on a FACS Fortessa (BD Biosciences).

Results were analyzed using FlowJo software.

in vitro generation of virus from PBMC or MDM

To generate PBMC origin HIV, PBMCs isolated and activated as described above were

infected with 2 × 106 RNA copies/mL NL4–3(AD8) for 24 hours. Cells were then washed 4

times in growth medium to remove the input viral stock and incubated for 4 days (approxi-

mately two full virus cycles) for a total infection time of 5 days. Virus containing supernatant

was collected, centrifuged at 300 g for 5 minutes then filtered through a 0.45 micron syringe fil-

ter (GVS) to remove cells and cellular debris. The number of virus genomes in the virus stock

was determined using the RealTime HIV-1 viral load test (Molecular Diagnostic Services). To

generate monocyte-derived macrophage virus, CD14+ monocytes were isolated and differenti-

ated as described above. Cells were then infected with with 2 × 107 RNA copies/mL NL4–3

(AD8) for 24 hours. Cells were washed 6 times with RPMI to remove input virus, and growth

media was replaced. Half the volume of media was replaced every 3 days for 14 days for a total

infection time of 15 days. Virus containing supernatant was collected, centrifuged and filtered,

and viral load determined as for PBMC virus. We used 7 different donors blood donors: 6009,

6013, 6017, 6019, 6026, 6033 and 6049.

Host cell marker detection on virion surface

The following protocol was adapted from the μMACS Streptavidin Kit protocol (Miltenyi): 1

μg of biotinylated antibodies to CD26 or CD36 (Ancell) were added to 1 mL of virus, mixed

and incubated for 30 min at room temperature. Next, 30 μL of strepavidin MicroBeads (Milte-

nyi) were added per sample, mixed and incubated at room temperature for 10 min. The sam-

ples were then loaded onto a μColumn, washed three times and bound virus eluted. Clinical

virus samples were centrifuged for 13,000 g for 30 seconds to clear debris before addition of

antibodies. To avoid overloading columns, in vitro generated virus stocks from either PBMCs

or macrophages were diluted to approximately 104 RNA copies/mL in PBS before incubation

with antibodies. We used the 7 donors above. Virus was precipitated once for six of the donors,

whereas 6049 was done in duplicate. The number of virus genomes in elutions from μColumns

was determined using the RealTime HIV-1 viral load test (Molecular Diagnostic Services). Out

of 22 CSF escape samples, 11 had sufficient volume for the assay (2 mL). One sample which

showed neither detectable CD26 nor CD36 was excluded from the analysis due to possible deg-

radation of the virus.

Generation of YFP-NL4–3(AD8)

pNL4–3(AD8) was used as the source of the macrophage tropic HIV ENV which was excised

from pNL4–3(AD8) using BamHI and EcoRI restriction enzymes (NEB) and ligated using T4

ligase (Invitrogen) into the pNL4–3-YFP vector (gift from David Levy), replacing the NL4–3

X4 specific HIV envelope gene between the unique EcoRI-BamHI restriction sites.

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Detection of ART concentrations in CSF and matched plasma by LC-MS/MS

Sample analysis was performed using an Agilent High Pressure Liquid Chromatography

(HPLC) system coupled to the AB Sciex 5500, triple quadrupole mass spectrometer equipped

with an electrospray ionization (ESI) TurboIonSpray source. The LC-MS/MS method was

developed and optimised for the quantitation of tenofovir, emtricitabine, efavirenz, lopinavir,

ritonavir, nevirapine, zidovudine, lamivudine, abacavir, atazanvir and dolutegravir in the same

sample. A protein precipitation extraction method using acetonitrile was used to process clini-

cal plasma and CSF samples. The procedure was performed using 50 μL of plasma or CSF. 50

μL of water and 50 μL of ISTD solution was added and the sample was briefly mixed. 150 μL of

acetonitrile was subsequently added to facilitate protein precipitation, vortex mixed and cen-

trifuged at 16000 g for 10 min at 4˚C. 170 μL of the clear supernatant was then transferred to a

clean micro-centrifuge tube and dried down using a SpeedVac dryer set at 40˚C. The dried

samples were then reconstituted in 100 μL of 0.02% sodium deoxycholate (Sigma) in Millipore

filtered water, vortex mixed, briefly centrifuged, placed in a small insert vial, capped, placed in

the auto sampler compartment (maintained at 4˚C) and analyzed using LC-MS/MS. The ana-

lytes were separated on an Agilent Zorbax Eclipse Plus C18 HPLC column using gradient elu-

tion. The column oven was set at 40˚C, a sample volume of 2 μL was injected and the flow rate

was set to 0.2 mL/min. Mobile phase A consisted of water with 0.1% formic acid and B con-

sisted of acetonitrile with 0.1% formic acid. The drug analytes were monitored using multiple-

reaction monitoring mode for positive ions except for efavirenz which was monitored in the

negative ion scan mode. Analyst software, version 1.6.2 was used for quantitative data analysis.

Blanked values for EFV, FTC and TFV were in the range of 3 ng/mL and this was set as the

detection limit.

Results

Prevalence of CSF escape

We sampled CSF and matched blood from participants living with HIV (n = 156) clinically

indicated for lumbar puncture as part of their diagnostic workup in Durban, South Africa

(Table 1). The majority (n = 80 or 51%) of participants showed an undetectable HIV viral load

Table 1. Participant details.

Infection state n

(F/M)

Age

(IQR)

VL CSF

(IQR)

VL Plasma

(IQR)

CD4 count

(IQR)

Years ART

(IQR)

Fraction co-infect Fraction pleocytosis1

CSF escape 22 40 2720 < 40 474 4.8 0.27 0.45

(11/11) (34–46) (897–28380) (344–585) (1.3–7.2)

Suppressed 58 34 < 40 < 40 509 2.3 0.33 0.33

(37/21) (29–42) (193–724) (0.9–6.1)

Viremic (total) 76 33 3680 14956 246 1.02 0.38 0.41

(48/28) (28–40) (173–26210) (699–70672) (91–411) (0.08–5)

Viremic (naïve) 34 35 9819 44902 324 N/A 0.32 0.53

(20/14) (30–44) (3255–78440) (13935–240120) (104–493)

Viremic (ART) 42 32 1280 1950 219 1.0 0.43 0.31

(28/14) (27–36) (< 40–5624) (245–31754) (52–379) (0.08–5)

VL in HIV RNA copies/mL blood. Limit of detection 40 copies/mL. 1Defined as >5 leukocytes per μL of CSF. 2Of viremic participants on ART.

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(VL) in the blood. Out of the subgroup of successfully ART suppressed individuals in the

blood, 22 (28% of those with an undetectable VL in the blood) had detectable viremia in the

CSF (CSF escape). The remainder of the participants (n = 76 or 49% of the total) had detectable

blood viremia either because they were treatment naïve (n = 34 or 45% of the viremic group)

or failing ART (n = 42 or 55% of the viremic group). We note that the CSF escape detected in

this study is likely neurosymptomatic CSF escape, leading to clinically indicated lumbar punc-

ture (see S1 Table for indications). This form of CSF escape may have a different cellular

source and infection dynamics relative to asymptomatic CSF escape [59].

The CSF escape group was significantly older compared to the suppressed group (p = 0.018,

Mann-Whitney U test). Otherwise, the CSF escape group was very similar to the suppressed

group, except for the VL in the CSF. The groups did not differ in either median CD4 count

(p = 0.78, Mann-Whitney), years on ART (p = 0.25, Mann-Whitney), fraction of individuals

with diagnosed co-infections (p = 0.78, Fisher’s exact test) or the ratio between males and

females (p = 0.79, Fisher’s exact test). Pleocytosis, abnormally high white blood cell concentra-

tions in the CSF usually indicating co-infection with another pathogen in the CNS, occurred

in 45% of CSF escape participants and 33% of suppressed participants. However, the trend of

higher pleocytosis in CSF escape was not statistically significant (p = 0.31, Fisher’s exact test).

Surprisingly, median CSF VL in CSF escape participants was not significantly different from

that of viremic participants (p = 0.96, Mann-Whitney). However, when compared to the treat-

ment naïve and ART experienced viremic participant subgroups separately, the CSF escape

participants showed a significantly lower median CSF VL compared to viremic naïve

(p = 0.022, Mann-Whitney) and significantly higher VL compared to viremic participants on

ART (p = 0.039, Mann-Whitney).

To exclude the confounding effect of treatment regimen [16], we concentrated on samples

from suppressed or CSF escape participants on a regimen of EFV, FTC, and TFV (first line

therapy at the time of sample collection), and on participants where sufficient sample volume

was available, for subsequent analysis.

CD26 expression differentiates between T cells and macrophages and

microglia

Previous work detecting host cell markers on the virion surface [46–50] converged on CD26

(T cell) and CD36 (macrophage lineage) host cell markers on the surface of the HIV virion as

the most robust markers for differentiation between cell types using this approach. We there-

fore proceeded to test whether these markers could indeed differentiate T cells from macro-

phages and microglia in our study population (Fig 1). We detected CD26 and CD36 on: 1)

CD3+ T cells from peripheral blood mononuclear cells (PBMC); 2) CD68+ monocyte derived

macrophages (MDM) (Fig 1A, see S1 Fig for gating strategy); 3) CD4+ T cells from lymph

nodes (LN); 4) CD68+HLA-DR+ macrophages from LN (Fig 1B, see S1 Fig for gating strategy

for LN cells); 5) primary human CD11b+P2RY12+ microglia from discarded neurosurgical tis-

sue (Fig 1C).

The level of CD26 showed a clear demarcation between T cells in the two compartments

and macrophages and microglia (Fig 1D, left panel). While there were differences in CD26

expression between T cells in PBMC and LN (37% versus 13% positive cells), MDM, LN mac-

rophages, and microglia had negligible or undetectable CD26 levels. In contrast, it was less

clear that CD36 could differentiate cell types in the South African population studied here (Fig

1D, right panel). While LN T cell showed negligible levels, CD36 expression in PBMC in our

study population was substantial and within the range of expression in macrophages and

microglia (Fig 1D, right panel). Since only about one quarter of T cells are positive for CD26,

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Fig 1. Expression of CD26 and CD36 surface markers on T cells and macrophages. (A) Flow cytometry detection of CD36 and CD26 on the surface of

CD3+ gated PBMC (left) or CD68+ monocyte derived macrophages (MDM) (right). (B) CD26 and CD36 detection on lymph node (LN) origin live CD45

+CD19-CD3+CD4+ T cells (left) or live CD45+CD3-CD19-CD68+HLA-DR+ macrophages (right). Staining was performed on three LN from the field of

indicated surgery of study participants 024–09-0233, 024–09-0274, and 024–09-0257. Shown is a representative result (participant 024–09-0257). (C)

primary human CD11bmidP2RY12+ microglial cells. (D) Violin plots of the fraction of cells expressing CD26 (left) and CD36 (right) of CD4 T cells from

PBMC (n = 12) or LN (n = 3), MDM (n = 6), lymph node macrophages (LN Mϕ, n = 3)), or microglia (n = 2). Number above each plot indicates median

percent positive cells.

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cells that are negative for CD26 can either be T cells or macrophages/microglia. However,

CD26 positives cells are not macrophages/microglia.

Given that CD36 in our assays could not discriminate well between T cells and macro-

phages/microglia, we have focused our analysis on CD26 level as an indicator of T cell origin.

Host cell markers on the virion surface are consistent with presence of T

cell origin HIV

To detect cellular origin of CSF HIV using CD26, we measured the ratio of the number of viri-

ons expressing CD26 on their surface to the total number of virions in the sample. Both quan-

tities were measured using a viral load (VL) assay as follows. We coupled CD26 antibodies to

magnetic beads and added the sample containing either virus from CSF or plasma samples, or

in vitro produced virus to a column containing the bound antibodies. We performed a VL

assay to determine: 1) the number of CD26 antibody bound virions after washing off unbound

virions, and 2) the VL of the virus suspension added to the column (Fig 2A). We normalized

by the VL of the virus added to the columns so that the measurement would not be affected by

the absolute VL, which varied between study participants. We infected either monocyte

derived macrophages (MDM) or peripheral blood mononuclear cells (PBMC) with the macro-

phage tropic HIV strain NL4–3(AD8), which infects both CD4 T cells and macrophages, to

test whether CD26 level can differentiate between the virions produced in each cell type using

this approach. Using YFP labelled macrophage tropic HIV (YFP-NL4–3(AD8)), we observed

no infection of CD14+ monocytes in PBMC (S2 Fig), indicating that the PBMC derived virus

originated in T cells. There was approximately an order of magnitude increase in the CD26

level normalized by input VL (p< 0.05, Kruskal-Wallis test with Dunn multiple comparison

correction) in virus originating from PBMC relative to MDM (Fig 2B, see S3 Fig for raw VL).

Similarly to what we observed using CD26 and CD36 in cell surface labelling, CD26 on the

viral surface could specifically differentiate between T cells and macrophages as the viral

source, but CD36 could not (Fig 2B and S3 Fig).

We next used the CD26 normalized by input VL to infer the cellular origin of in vivo derived CSF escape virus for CSF samples with sufficient volume (11 samples excluded due to

insufficient volume (2 mL minimum) for the assay and one excluded based on poor sample

quality (Materials and methods)). We compared the CD26/input ratio obtained from CSF

escape HIV with the in vitro values for MDM and PBMC (Fig 2B). The median CSF escape

CD26 level was significantly higher than the ratio obtained from HIV derived from MDM

infection (p-value ���< 0.001, Kruskal-Wallis test with Dunn multiple comparison correc-

tion). In contrast, it was similar to the CD26 level in T cell derived virus from in vitro infected

PBMC (p> 0.99, using the same test).

If the number of virions bound to the anti-CD26 antibodies from the CSF is very small, it

may be difficult to accurately estimate the ratio of virions expressing CD26 relative to input.

We therefore also examined the absolute numbers of anti-CD26 bound and input virions (S4

Fig). To ensure that the range of observed values is taken into account, we calculated the geo-

metric mean of the data. We observed that even the CSF with the lowest number of viral copies

measured was approximately an order of magnitude above the detection limit of the VL assay

of 40 HIV RNA copies/mL. The geometric mean of the number of CD26 expressing virions, at

about 1600 copies/mL, was approximately 4-fold lower than the total number of virions in the

sample. Such a decrease would be expected even if all the virus was produced in T cells, since

we observed that only about one quarter of T cells express detectable CD26 (Fig 1). Given that

the number of CD26 expressing virions was neither at the assay detection limit nor a small

proportion of the total virions in the sample, it is unlikely that our estimate of the ratio of

PLOS PATHOGENS T cell derived HIV-1 in the CSF in the face of suppressive antiretroviral therapy

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009871 September 23, 2021 9 / 21

CD26 expressing virions in the total virus pool is strongly affected by these factors. We there-

fore conclude that the measurement of CD26 on HIV from the CSF of individuals with CSF

escape is consistent with the presence of T cell origin virus.

We also investigated origin of CSF and plasma virus from plasma viremic participants

(Fig 3). There was no significant trend to higher or lower CD26 level between the matched

CSF and plasma compartments from the same participant (Fig 3A). The median CD26/input

ratio from CSF virus of participants with viremia was significantly different from that of

MDM generated virus and consistent with the presence of T cell origin virus. However, there

was a wide range of CD26 levels between participants, including low CD26 levels similar to

virus produced in vitro by MDM (Fig 3B). In plasma of viremic participants, the range of

CD26 levels was large and overlapped CD26 expression from both T cell and MDM derived

virus (Fig 3B).

Fig 2. HIV from CSF escape contains the CD26 host surface marker consistent with T cell origin. (A) Schematic of method. Cell-free virus was bound to columns

with anti-CD26 antibodies linked to magnetic beads. After washing off unbound virus, virus bound to the columns was eluted and quantified using a viral load assay.

(B) Monocyte derived macrophages or peripheral blood mononuclear cells were infected with NL4–3(AD8) macrophage tropic HIV and supernatants from infected

cells were loaded on columns with CD26 antibody and normalized by viral input. Results were compared with virus derived from study participants with CSF escape.

Shown are median and IQR of supernatants derived from 10 CSF escape participants or in vitro infections of PBMC or MDM from 7 healthy blood donors, where

blood from one of the donors was used twice (n = 8 total experiments). p-values are � < 0.05; ��� < 0.001 as determined by Kruskal-Wallis test with Dunn multiple

comparison correction.

https://doi.org/10.1371/journal.ppat.1009871.g002

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CSF ART levels similar between individuals with CSF escape and full

suppression

To investigate whether CSF escape is due to lowered CSF ART levels, we measured the plasma

and CSF concentrations of ART regimen components using liquid chromatography–tandem

mass spectrometry (LC-MS/MS). We compared drug concentrations between viremic partici-

pants on ART, suppressed participants, and CSF escape participants. Confirming previous

results [19, 60, 61], we detected a sharp drop in EFV concentrations in the CSF relative to

plasma (Fig 4), with EFV about two orders of magnitude lower. The decline in FTC levels

between plasma and CSF was much more attenuated, showing FTC has good penetration into

the CSF. TFV levels were close to or below the limit of detection in the CSF for most partici-

pants, and about an order of magnitude higher in the plasma.

We compared drug levels between groups, and included participants reported to be treat-

ment naïve (Fig 5). We observed that, as expected, the treatment naïve group had drug levels

below the threshold of detection. Viremic participants showed a bimodal distribution of drug

levels in the plasma and CSF, likely corresponding to two subgroups: individuals either failing

therapy or who are non-adherent. There was no significant difference between levels of EFV

between suppressed and CSF escape participants in either the plasma or CSF (p = 0.83 and

p> 0.99 for plasma and CSF respectively, Kruskal-Wallis test with Dunn multiple comparison

correction). The same was true of FTC (p> 0.99 and p> 0.99 for plasma and CSF respec-

tively) and TFV (p> 0.99 and p> 0.99 respectively). This indicates that there is no reduction

in CSF drug levels in CSF escape individuals relative to individuals with full suppression.

Fig 3. in vivo cellular source of HIV from viremic participants. (A) CD26/input ratio in participant matched CSF and plasma from viremic participants where both

were detectable (n = 32). (B) CD26/input ratio for all available samples (n = 34 for CSF, n = 48 plasma) from CSF and plasma of viremic study participants compared

to virus derived from in vitro infected MDM and T cells. p-values are � < 0.05; �� < 0.01 as determined by the Kruskal-Wallis test with Dunn multiple comparison

correction.

https://doi.org/10.1371/journal.ppat.1009871.g003

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Drug resistance mutations not essential for a detectable viral load in the

CSF

One possible explanation for CSF escape is accumulation of drug resistance mutations

(DRMs) from previous gaps in adherence or other factors. We therefore sequenced CSF virus.

In participant samples where CSF virus sequencing was successful (Table 2), we observed

DRMs including the M184V high level resistance mutation to FTC and the L100I, K103N,

V106M, and G190A mutations to EFV (https://hivdb.stanford.edu/).

Fig 4. Sharp drop in EFV levels between plasma and CSF. Medians and IQR of EFV, FTC, and TFV concentrations in the plasma versus CSF in participants

who were viremic on ART (plasma n = 32, CSF n = 31), suppressed (plasma and CSF n = 48), or showed CSF escape (plasma n = 19, CSF n = 18). Horizontal

dotted line indicates limit of detection (3 ng/mL). p-values are: �< 0.05; ��< 0.01; ����< 0.0001 using the Mann-Whitney U test.

https://doi.org/10.1371/journal.ppat.1009871.g004

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PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009871 September 23, 2021 12 / 21

Fig 5. ART concentrations in plasma and CSF are similar in individuals with full suppression and CSF escape. Medians and IQR of EFV, FTC, and TFV

concentrations in the plasma (left) and CSF (right) in individuals who were viremic on ART (plasma n = 32, CSF n = 31), suppressed (plasma and CSF n = 48),

showed CSF escape (plasma n = 19, CSF n = 18), or were reported as treatment naïve (plasma n = 33, CSF n = 31). Horizontal dotted line indicates limit of

detection (3 ng/mL). p-values are: �< 0.05; ��< 0.01; ����< 0.0001 using the Kruskal-Wallis test with Dunn multiple comparison correction.

https://doi.org/10.1371/journal.ppat.1009871.g005

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PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009871 September 23, 2021 13 / 21

One participant (PID 0172) showed no detectable ART and, as expected, showed no CSF

DRMs. HIV from one of two participants with CSF escape also contained DRMs (PID 0213),

while HIV sequenced from the second participant (PID 0161) did not. The CSF escape partici-

pant with detected CSF DRMs was on a lopinavir/ritonavir protease inhibitor based therapy as

detected by LC-MS/MS. DRMs included M184V, V106M, and G190A, the latter mutations a

possible result of first line EFV regimen failure. In contrast, PID 0161, who was on EFV based

therapy, had no CSF DRMs detected. Two additional participants (PID 0148 and 0164)

showed CSF discordance, defined as a CSF VL about 0.5 log10 or greater of plasma VL [11].

Both were on EFV based therapy. EFV and FTC CSF concentrations in these participants were

at or above the median CSF concentrations for suppressed and CSF escape participants

(Table 2). Yet only the K101E DRM, conferring low level resistance to EFV (https://hivdb.

stanford.edu/) was detected. An additional participant (PID 0168) on an EFV based regimen

showed a CSF viral load. The level of EFV in the CSF was near background for this participant.

However, FTC was above the median concentration for suppressed and CSF escape partici-

pants. No DRMS were detected in CSF virus from this participant. These results may indicate

that DRMs may not be necessary for a detectable VL in the CSF in the face of ART.

Discussion

Here, we examined the cellular source of viremia in study participants with CSF escape in Dur-

ban, South Africa, and whether drug levels in the CSF are lower in individuals with CSF escape

versus those suppressed both in the blood and CSF. Detection of the CD26 T cell host surface

marker on the viral envelope was consistent with the presence of at least some T cell origin

virus in our study population, where the overwhelming majority of people living with HIV are

infected with clade C virus [62]. Our conclusion that CD26 was effective in differentiating T

cells from myeloid lineage cells was based on the observation that neither monocyte derived

macrophages, nor primary human macrophages from human lymph nodes, nor human

microglia, expressed CD26. Since only a fraction of T cells were positive for CD26, cells that

were negative for CD26 could either be T cells or macrophages or microglia. However, expres-

sion of CD26 was only on T cells.

Consistent with this, HIV produced from in vitro infection of macrophages showed no

CD26 expression. Given that CD26 is absent in myeloid lineage cells, a reasonable conclusion

is that these cells did not generate virus with the CD26 host marker on its surface. This does

not exclude the presence of virus produced in myeloid lineage cells in CSF escape, since about

Table 2. Drug resistance mutations in CSF.

PID VL (copies/ml)

Pl/CSF

Drug resistance mutation TFV (ng/ml)

Pl/CSF

FTC (ng/ml)

Pl/CSF

EFV (ng/ml)

Pl/CSF

LPV (ng/ml)

Pl/CSF

RTV (ng/ml)

Pl/CSF

0161 <40/1350 None detected 76/3.0 445/256 1490/4.4 3.0/3.0 3.0/3.0

0213 <40/1330 V82A/D67N/K70E/L74I/V106M/M184V/G190A 225/4.9 420/153 3.0/3.0 7730/7.0 935/3.0

0189 12802/310 K65R/V75I/M184V/L100I/K103N/P225H 82/3.0 146/175 1990/9.6 3.0/3.0 3.0/3.0

0133 19551/28250 M46I/V82A/D67N/K70R/M184V/T215F/K103N/K238T 19/5.0 63/71 3.0/3.0 14/3.0 3.0/3.0

0183 58000/1300 D67N/M184V/K103N/V106M 55/3.0 229/129 2460/3.0 3.0/3.0 3.0/3.0

0140 254/21370 K101E 33/3.0 98/123 5530/40 3.0/3.0 3.0/3.0

0164 620/2900 K101E 43/3.0 518/169 4130/8.8 3.0/3.0 3.0/3.0

0168 936/3030 None detected 56/3.0 286/196 2370/3.5 3.0/3.0 3.0/3.0

0172 24000/801 None detected 3.0/3.0 3.0/3.0 3.8/3.0 3.0/3.0 3.0/3.0

PID: participant ID. Pl: plasma. LPV: lopinavir. RTV: ritonavir. Limit of detection 40 copies/mL for VL and 3.0 ng/mL for antiretroviral drugs.

https://doi.org/10.1371/journal.ppat.1009871.t002

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three-quarters of virions in the CSF did not express CD26 (S4 Fig), either because it was pro-

duced in myeloid lineage cells or because it was produced in T cells which did not express

CD26.

Our results support the previously documented low levels of CD26 expression on macro-

phage origin HIV [46]. We did not find that CD36 was a robust marker to differentiate T cells

from macrophages in our study population. We detected appreciable levels of CD36 on T cells,

consistent with a recent study [63] but not two others [46, 64]. This was consistent with our in vitro infection results of T cells and MDM: Both T cell and MDM produced virus expressed

CD36 at similar levels but MDM generated virus could be differentiated from T cell generated

virus by the absence of CD26. Reasons for differences could include the specific study popula-

tion, given the role of CD36 in fatty acid uptake [65]. T cell and macrophage surface marker

detection in our study population was therefore critical in the choice of which marker to

analyze.

To our knowledge, this is the first time CSF escape has been examined in Sub-Saharan

Africa in a relatively large number of study participants. The CSF escape detected in this study

was likely neurosymptomatic [59], and asymptomatic CSF escape may have a different mecha-

nism of cellular persistence. The fraction of study participants with CSF escape, at 28%, was

high relative to that found in other studies [11–16, 25]. This may be partly explained by the

presence of co-infections and the selection of neurosymptomatic cases but may also be related

to the HIV clade C subtype.

Infections are the most common cause of pleocytosis, the elevation of white blood cell num-

bers in the CSF [66]. Pleocytosis occurred in about half of participants with CSF escape,

although the frequency was not significantly different from the suppressed group. Therefore,

HIV could be produced in other cell types such as macrophages or microglia, then be ampli-

fied by the CD4+ T cells in the CSF. Our data does not exclude this possibility, it only indicates

that some of the virions have a T cell origin. The data was also consistent with T cells produc-

ing HIV in the CSF in viremic individuals.

We did not observe lower ART levels in the CSF of CSF escape participants. CSF virus from

two successfully sequenced CSF escape participants in our study showed DRMs in the partici-

pant on a protease based ART regimen, as determined by LC-MS/MS, but not in the partici-

pant on the first line EFV based regimen. Two additional participants on EFV based therapy

showed CSF discordance, defined as a CSF VL about 0.5 log10 or greater of plasma VL [11].

CSF virus from both showed only low level resistance to EFV. With the limitation that the

number of sequenced viruses was small, this may indicate that DRMs are not always essential

for CSF escape or CSF discordance if the regimen is based on EFV. CSF escape despite the

maintenance of suppressive ART and lack of drug resistance may be consistent with reactiva-

tion from latency [67, 68], more efficient HIV replication due to HIV cell-to-cell spread [58,

69–71], or both.

T cell origin HIV in CSF escape has been described in a recent study where two out of three

participants with asymptomatic CSF escape had T cell tropic virus which was unlikely to utilize

the low levels of CD4 expression found on macrophages [25]. This study was performed in a

different population from that described in our study, and used techniques complimentary

ours to arrive at similar conclusions. Infected T cells may therefore be present in the CSF in

the face of ART, including in the Sub-Saharan African population most affected by HIV

infection.

The observation that some T cells are infected and produce virus in the CSF is unexpected

and may have important implications. Mechanisms driving such infections can include T cell

infection by CNS resident cell types such as macrophages, ongoing infection between CNS res-

ident CD4 T cells, or trafficking of infected T cells into the CSF from other compartments and

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PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009871 September 23, 2021 15 / 21

potentially subsequent reactivation from latency. Unlike microglia and perivascular macro-

phages, which are compartmentalized to the CNS, T cells may circulate from and to other

compartments. In addition, T cells may form a latent reservoir [72–77]. Given that potential

curative approaches would likely need to eradicate all reservoirs [67, 68], the cellular nature of

the CNS reservoir must be determined for these approaches to succeed.

Supporting information

S1 Fig. Gating strategy. (A) Gating for PBMC T cells (left two panels) and MDM (right two

panels). Cells were stained with APC conjugated anti-CD3 (PBMC) or anti-CD68 (MDM)

antibodies. (B) Gating for lymph node T cells. (C) Gating for lymph node macrophages. Repre-

sentative result from one of 3 LN donors (024–09-0257).

(TIF)

S2 Fig. No HIV infection of PBMC origin CD14+ cells. PBMC were infected with 2 × 107

RNA copies/mL YFP-NL4–3(AD8). 2 days post-infection, cells were collected and stained

with CD3 and CD14 antibodies, then analyzed for infection by detection of YFP positive cells

in the CD3+ and CD14+ populations using flow cytometry. Shown are median and IQR for

different blood donors. The median fraction of infected CD3+ gated CD4+ PBMC was 2.4%

(IQR 1.7–3.2). No infected CD14+ monocytes were detected. The difference was significant

(p-value is ����< 0.0001; Mann-Whitney U test). Brown circles denote blood donor 0020, red

circles donor 0019, green donor 0049, and blue donor 0051.

(TIF)

S3 Fig. Absolute HIV RNA copies from CD26 versus CD36 columns in in vitro infection.

MDM or PBMCs were infected with NL4–3(AD8) macrophage tropic HIV able to infect both

cell types. Supernatant from the infected cells was collected and diluted to 104 HIV RNA cop-

ies/mL. Half the diluted supernatant was applied to CD26 binding columns and the other half

to CD36 binding columns to quantify the number of CD26 and CD36 expressing virions.

Shown are median and IQR for different blood donors. Macrophage values: CD26 median 208

HIV RNA copies/mL (85–440 copies/mL), CD36 median 5403 copies/mL (3076–9263 copies/

mL). PBMC values: CD26 median 6600 copies/mL (5749–11185 copies/mL), CD36 median

5862 copies/mL (3309–5862 copies/mL). p-values are: �< 0.05; ��< 0.01; ����< 0.0001 by

Kruskal-Wallis non-parametric test with Dunn multiple comparisons correction.

(TIF)

S4 Fig. Absolute HIV RNA copies per milliliter of CD26 expressing versus total virus in

CSF escape samples. A viral load assay was performed on the CSF escape samples. Samples

were then added to a column with anti-CD26 bead bound antibodies for immuno-capture of

virus expressing CD26. Captured virus was then eluted and viral load assay performed. Red

dotted line represents limit of assay detection. GM: geometric mean of n = 10 participants for

each condition. Geometric mean was 5874 HIV RNA copies/mL for total virus, and 1591 cop-

ies/mL for virus with CD26 surface expression.

(TIF)

S1 Table. Participant indications for lumbar puncture.

(XLSX)

Author Contributions

Conceptualization: Gila Lustig, Alex Sigal.

PLOS PATHOGENS T cell derived HIV-1 in the CSF in the face of suppressive antiretroviral therapy

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009871 September 23, 2021 16 / 21

Data curation: Gila Lustig, Bernadett I. Gosnell, Mahomed-Yunus S. Moosa, Suzaan Marais,

Prakash M. Jeena, Rohen Harrichandparsad, Vinod B. Patel, Alex Sigal.

Formal analysis: Gila Lustig, Alex Sigal.

Funding acquisition: Alex Sigal.

Investigation: Gila Lustig, Sandile Cele, Farina Karim, Anne Derache, Abigail Ngoepe, Kha-

dija Khan, Ntombi Ntshuba, Suzaan Marais, Prakash M. Jeena, Katya Govender, John

Adamson, Henrik Kløverpris, Rohen Harrichandparsad.

Methodology: Gila Lustig, Sandile Cele, Anne Derache, Khadija Khan, Katya Govender, John

Adamson, Ravindra K. Gupta, Alex Sigal.

Project administration: Farina Karim, Alex Sigal.

Supervision: Mahomed-Yunus S. Moosa, Henrik Kløverpris, Ravindra K. Gupta, Vinod B.

Patel, Alex Sigal.

Validation: Bernadett I. Gosnell, Suzaan Marais, Alex Sigal.

Visualization: Alex Sigal.

Writing – original draft: Gila Lustig, Mahomed-Yunus S. Moosa, Alex Sigal.

Writing – review & editing: Gila Lustig, Alex Sigal.

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