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Impact of analytical treatment interruption on the central nervous system in a simian-HIV model

Hsu, Denise C.a,b; Silsorn, Dechaa; Inthawong, Dutsadeea; Kuncharin, Yanina; Sopanaporn, Jumpola; Im-Erbsin, Rawiwanc; Chumpolkulwong, Kesarac; O’connell, Robert J.a; Michael, Nelson L.d; Ege, Christine A.c; Vasan, Sandhyaa,b

doi: 10.1097/QAD.0000000000002270
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Objective(s): Analytical treatment interruption (ATI) studies are often used to evaluate potential HIV cure strategies. This study was conducted to determine the impact of ATI on simian-HIV (SHIV) infection in the central nervous system.

Design: Animal study.

Methods: Nine rhesus macaques were inoculated with SHIV-1157ipd3N4. Antiretroviral therapy (ART) was administered from week 2 to 18. At week 18, four animals were euthanized (no-ATI-group) and five underwent ATI (ATI-group) and were euthanized at 12 weeks post viral rebound. Plasma and cerebrospinal fluid (CSF) SHIV-RNA, markers of inflammation and brain CD3+, CD68+/CD163+ and RNA+ cells were measured.

Results: All nine animals were SHIV-infected, with median pre-ART plasma and CSF SHIV-RNA of 6.2 and 3.6 log10copies/ml. Plasma and CSF IL-15, monocyte chemoattractant protein-1, IFN-γ-induced protein-10 and neopterin increased postinfection. ART initiation was associated with rapid and complete suppression of plasma viremia and reductions in plasma and CSF IL-15, IFN-γ-induced protein-10, neopterin and CSF monocyte chemoattractant protein-1. Median time to plasma viral rebound was 21 days post-ATI. At 12 weeks postrebound, CSF SHIV-RNA was undetectable and no increases in plasma and CSF markers of inflammation were found. Higher numbers of CD3+ and CD68+/CD163+ cells were seen in the brains of 3/5 and 1/5 animals, respectively, in the ATI-group when compared with no-ATI-group. SHIV-RNA+ cells were not identified in the brain in either group post-ATI.

Conclusion: ATI in macaques that initiated ART during early SHIV-1157ipd3N4 infection was associated with mild, localized T-cell infiltrate in the brain without detectable SHIV-RNA in the brain or CSF, or elevation in CSF soluble markers of inflammation.

aU.S. Military HIV Research Program, Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand

bHenry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA

cDepartment of Veterinary Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand

dU.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.

Correspondence to Dr Denise C. Hsu, Department of Retrovirology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand. Tel: +66 2 696 2700x4905; e-mail: hsudc@hiv-th.org

Received 14 May, 2018

Accepted 30 April, 2019

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Introduction

Antiretroviral therapy (ART) for the treatment of HIV infection is currently lifelong due to the persistence of the latent viral reservoir [1,2]. Because sensitive, accurate and precise quantification of the latent viral reservoir remains elusive [3], analytical treatment interruption (ATI) is often used to evaluate the efficacy of interventions on reducing the viral reservoir and achieving HIV remission [4]. Knowledge gaps exist on how best to conduct ATI studies and potential risks involved [5], especially in the central nervous system (CNS), where virus may be compartmentalized [6–8] and ART penetration may be variable [9–11].

In HIV-infected individuals, even low levels of cerebrospinal fluid (CSF) viremia (<100 copies/ml in untreated primary HIV infection [12] and less than 40 copies/ml on conventional assay on ART with concomitant plasma viral suppression [13]) are associated with elevated neopterin, a macrophage and microglial activation marker associated with CNS immune activation [14]. Despite ART with plasma and CSF viral suppression, individuals with neurocognitive impairment had elevated CSF neopterin [15]. Both ‘Boston Patients’, HIV-infected men with no detectable HIV-DNA or HIV-RNA in circulating CD4+ T cells or plasma, while on ART, after allogeneic hematopoietic stem-cell transplantation; had detectable HIV-RNA in CSF on viral rebound after ATI. One also had nausea, vomiting, headache, fevers and low-grade lymphocytic pleocytosis in the CSF consistent with HIV-associated meningitis [16]. Therefore, viral rebound during ATI may lead to systemic and/or localized CNS inflammation, that if not reversed, may potentially contribute to neurocognitive impairment. Finally, cure interventions may not penetrate the blood–brain barrier equally.

The use of nonhuman primate (NHP) models offer multiple advantages, including the ability to access brain tissues to quantify viral load, latent reservoir size and inflammation. A number of ATI studies have been performed in NHPs treated with ART after simian immunodeficiency virus (SIV) [17–53] or simian-HIV (SHIV) [54–60] infection. However, the majority focused on viral burden in the peripheral blood and rarely assessed impacts on the CNS. Therefore, there is a need to elucidate the sequelae of cure interventions and ATI on the CNS.

In two pigtailed macaques dual-inoculated with an immunosuppressive SIV strain (SIV/DeltaB670) and a neurovirulent molecular clone (SIV/17E-Fr) that received ART from days 12 to days 292 and 232 post inoculation, plasma viral rebound occurred 3 and 4 days post-ATI. SIV-RNA was detectable by PCR in brain (163 copies/mg) and CSF (149 copies/ml) and by in-situ hybridization (ISH) in the occipital cortex, in one animal that had intermittent viremia on ART due to drug resistance. The other animal had no detectable SIV-RNA by PCR or ISH in the brain [23]. In another study involving six SIV/DeltaB670 and SIV/17E-Fr dual-inoculated pigtailed macaques, initiated on ART 12 days post inoculation and underwent ATI after 3 months of viral suppression, plasma and CSF viral rebound occurred at a median of 4 and 12 days, respectively. Postrebound plasma and CSF setpoint viral load was 5–6 and 4 log10copies/ml, respectively. SIV-RNA was detectable by PCR in the spinal cord and brain in 5/6 and 1/6 animals. Soluble or cellular markers of inflammation in CSF or brain were not reported [61]. Increased brain glucose metabolism as measured by fluorodeoxyglucose uptake, which may be indicative of neuroinflammation, was seen 1-month post-ATI in five SIV-infected macaques; was positively correlated with plasma and CSF SIV-RNA and negatively correlated with peripheral blood CD4+ and CD8+ T-cell counts post-ATI [62]. Therefore, CNS viral rebound during ATI is highly variable, and may potentially be correlated with increased metabolic activity and neuroinflammation.

Many of the proposed potential therapies for HIV cure target the HIV envelope, necessitating SHIV expressing HIV envelope to model their effects in rhesus macaques. Previously, we demonstrated that SHIV-1157ipd3N4-infected rhesus macaques displayed viral kinetics that mimicked early HIV infection, with evidence of SHIV-infection and immune activation in the CNS [63]. We conducted the present study to determine the effect of ATI on the CNS.

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Methods

Animals were housed at the AAALAC International-accredited, Armed Forces Research Institute of Medical Science (AFRIMS; Bangkok, Thailand). The protocol was approved by the Institutional Animal Care and Use Committee. Research was conducted in compliance with Thai laws, the Animal Welfare Act and other US federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, 2011 edition [64].

Nine men, adult, Indian-origin rhesus macaque (Macaca mulatta), excluding major histocompatibility complex alleles restrictive for SIV (MamuA*01, B*08 and B*17), were inoculated intrarectally with SHIV-1157ipd3N4 (an R5-tropic, mucosally transmissible virus, constructed using SIVmac239 backbone, encoding an HIV subtype C env derived from a Zambian infant [65]), at 3.9 × 107 RNA copies, at week 0. Daily ART (9-[(R)-2-(phosphonomethoxy)propyl]adenine PMPA 20 mg/kg, emtricitabine 50 mg/kg and dolutegravir 2.5 mg/kg), was administered subcutaneously, from weeks 2 to 18. No-ATI-group animals (n = 4) were humanely euthanized at weeks 17 and 18 while on ART. ATI-group animals (n = 5) underwent ART cessation at week 18 and continued clinical and laboratory monitoring until humane euthanization 12 weeks post viral rebound, at weeks 33–36.

Plasma and CSF SHIV-RNA levels were measured using real-time quantitative PCR with a lower limit of detection of 10 copies/ml [63,66]. Plasma and CSF IL-15, monocyte chemoattractant protein (MCP)-1 levels were quantified using a customized multiplex cytokine kit (Merck KGaA, Darmstadt, Germany). Plasma and CSF IFN-γ-induced protein 10 (IP-10) and neopterin levels were quantified using ELISA, all using previously described methods [63].

To quantify brain cellular inflammatory infiltrate, paraformaldehyde (4%)-fixed, paraffin-embedded tissues from the posterior cingulate gyrus, at 5-μm thick, on slides were deparaffinized. RNAscope was performed using SIVmac239 probe and RNAscope 2.5 HD Detection Kit-BROWN (Advanced Cell Diagnostics, Newark, CA, USA) according to manufacturer's instructions as described by Deleage et al.[67]. Tyramide-cyanine 3.5 plus (PerkinElmer, Waltham, MA, USA) was used for fluorescence detection. Slides were then washed with Tris-buffered-saline with 0.1% Tween 20 (TBS-T), blocked with 5% goat serum and 5% bovine serum albumin in TBS-T, washed, and incubated with mouse anti-CD68 (clone KP1; Biocare Medical, Pacheco, CA, USA), mouse anti-CD163 (clone 10D6; Leica Biosystems Nussloch, Germany) and rabbit anti-CD3 (Clone SP7; Thermo Fisher Scientific, Waltham, MA, USA), overnight at 4 °C. Slides were washed; incubated with goat antimouse IgG Alexa Fluor 647 and goat antirabbit IgG Alexa Fluor 488 (Thermo Fisher), for 2 h at room temperature (RT); washed; incubated with Sudan Black solution (0.1%), for 15 min, RT; washed; counterstained with 4,6-diamidino-2-phenylindole (Thermo Fisher), 5 min, RT; washed and mounted with ProLong gold antifade mountant (Thermo Fisher Scientific). Images were captured using an Olympus FV10i confocal microscope. Slides were surveyed using a ×40 objective and positive staining was confirmed with 60× oil-immersion objective. For enumerating CD3+ and CD68+/CD163+ cells, a total of 80 fields from two noncontiguous sections were counted. Due to the rarity of RNA+ cells, a total of 120 fields from three noncontiguous sections were counted. Lymph node from a rhesus macaque, 29 weeks after SHIV-1157ipd3N4 inoculation, was used as positive control.

Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Comparisons of soluble markers of inflammation between time-points were performed using Wilcoxon matched-pairs signed rank tests. Comparisons of CD3+ and CD68+/CD163+ cells between ATI and no-ATI groups were performed using the Mann–Whitney test. All P values were two-sided, with a P value of less than 0.05 considered significant.

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Results

All nine animals were SHIV-infected, with median pre-ART (week 2) plasma and CSF SHIV-RNA of 6.2 (range 4.8–6.6) and 3.6 (range 1.7–4.1) log10copies/ml respectively (Fig. 1a and b). There was a significant increase in levels of plasma and CSF IL-15 (median 17.3 vs. 9.7 pg/ml, P = 0.004 and 20.0 vs. 11.6 pg/ml, P = 0.004), MCP-1 (median 215.4 vs. 151.8 pg/ml, P = 0.012 and 574.7 vs. 202.7 pg/ml, P = 0.004), IP-10 (median 267.0 vs. 80.0 pg/ml, P = 0.008 and 32.8 vs. 20 pg/ml, P = 0.004) and neopterin (median 21.5 vs. 7.5 nmol/l, P = 0.004 and 16.7 vs. 2.2 nmol/l, P = 0.004) at week 2, when compared with preinfection (Fig. 1c–f).

Fig. 1

Fig. 1

ART initiation was associated with rapid and complete suppression of plasma viremia (Fig. 1a and b). There were reductions in plasma and CSF IL-15 (to median 7.3 pg/ml, P = 0.004 and 11.4 pg/ml, P = 0.004), CSF MCP-1 (to median 167.4 pg/ml, P = 0.004), plasma and CSF IP-10 (to median 44.0 pg/ml, P = 0.008 and 20.0 pg/ml, P = 0.004) and neopterin (to median 8.0 nmol/l, P = 0.004 and 2.1 nmol/l, P = 0.004) at week 18 when compared with week 2.

All animals in the ATI-group had undetectable plasma SHIV-RNA on ART cessation at week 18. Plasma viral rebound occurred at a median of 21 (range 17–28) days post-ATI, with median postrebound peak and setpoint SHIV-RNA of 1890 (range 355–19900) copies/ml and 53 (range 10–204) copies/ml. Viral rebound dynamics varied among the five animals. The two animals with the earliest plasma viral rebound had relatively higher peak postrebound viremia while another animal had reduction in viral RNA to the level of limit of detection for two weeks before a second higher rebound. CSF SHIV-RNA was undetectable in both groups at the time of euthanasia. At 12 weeks postrebound, when compared with pre-ATI, there was no obvious increase in plasma and CSF IL-15, MCP-1, IP-10 and neopterin levels.

Immunofluorescence of brain tissues from the posterior cingulate gyrus showed higher numbers of CD3+ cells in 3/5 and CD68+/CD163+ cells in 1/5 animals in the ATI-group when compared with no-ATI-group (median 15 vs. 4 CD3+ cells and 9 vs. 9.5 CD68+/CD163+ cells per 80 fields, P = 0.23 and 0.95, respectively, Fig. 2). No RNAscope positive cells were identified in either group.

Fig. 2

Fig. 2

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Discussion

Neurologic studies of ATI in humans involve characterizing inflammation and viral levels in CSF but cannot directly assess effects on the brain. In eight individuals treated during Fiebig I acute HIV infection who underwent ATI, median time to plasma viral rebound was 26 days. At time of plasma viral rebound, CSF (available from 4/8 participants) showed HIV-RNA of 30, 78, 739 and 13 462 copies/ml. No changes in CSF and plasma MCP-1, IP-10, neopterin and soluble(s) CD14 levels and neuronal and inflammatory measures on magnetic resonance (MR) spectroscopy were identified [68]. Animal models that mimic these clinical characteristics are needed to more finely elucidate the effects of ATI in the brain.

In this study, SHIV-1157ipd3N4-infected rhesus macaques showed rapid and sustained viral suppression after ART initiation. The median time to viral rebound post-ATI was 21 days, similar to individuals who initiated ART during acute HIV-infection [69]. At 12 weeks post viral rebound, SHIV was undetectable in CSF and there was also no obvious increase in plasma and CSF markers of inflammation that were elevated during initial infection. Quantification of T cell and myeloid inflammatory infiltrate in the posterior cingulate gyrus revealed higher CD3+ cells in 3/5 and higher CD68+/CD163+ cells in only 1/5 animals in the ATI-group. This does not exclude the possibility of infiltration into other brain regions and is consistent with mild T-cell infiltration in brain previously reported in early untreated SHIV-1157ipd3N4 infection [63]. The focus on the posterior cingulate gyrus was based on the findings of elevated choline/creatine levels in this region on MR spectroscopy of individuals with acute HIV infection that also correlated with higher CSF neopterin [70]. The lack of significant increase in CSF soluble markers of inflammation despite mild T-cell brain infiltrate suggests that immune pertubations may be focal and localized rather than widespread and generalized. Because the clinical significance of this infiltration is unclear, studies are ongoing to correlate these changes to potential alterations in neurobehavior in rhesus macaques.

A number of animal models, can be utilized to probe different aspects of the impact of ATI on the CNS. Humanized mice allow the direct manipulation of various immune pathways to tease out pathogenesis [71]. SIV-infected NHPs display high pathogenicity and are thus well suited to assess more advanced disease. SHIV is less virulent, thus can model more subtle events prior to end-stage disease. The presence of HIV envelope further allows the evaluation of interventions that target the HIV envelope, including therapeutic vaccines and humanized monoclonal antibodies.

In summary, ATI in macaques that initiated ART during early SHIV-1157ipd3N4 infection was associated with mild, localized T-cell infiltrate in the brain without detectable SHIV-RNA in the brain or CSF, or elevation in CSF soluble markers of inflammation. Evaluation of the CNS should be incorporated into more ATI studies in animals to further understand the potential impact of ATI in the CNS in humans.

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Acknowledgements

The authors wish to thank Gilead Sciences, Inc and Viiv Healthcare for their generous donation of ART; Dr Ruth Ruprecht for provision of SHIV-1157ipd3N4; Drs Jake Estes and Claire Deleage for consultation on RNAscope technology; and Sujitra Tayamun for assistance with slide preparation for immunohistochemistry and RNAscope analyses.

Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted under an approved animal use protocol in an AAALACi accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.

The work was supported by a cooperative agreement (W81XWH-11-2-0174) between the Henry M. Jackson Foundation for the Advancement of Military Medicine Inc. and the U.S. Department of the Army.

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Conflicts of interest

There are no conflicts of interest.

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Keywords:

analytical treatment interruption; antiretroviral therapy; central nervous system; HIV infection; simian-HIV infection

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