Decay of HIV DNA in the Reservoir and the Impact of Short Treatment Interruption in Kenyan Infants

Decay of HIV DNA in the Reservoir and the Impact of Short Treatment Interruption in Kenyan Infants Open Forum Infectious Diseases BRIEF REPORT Few studies have measured the impact of TI on HIV reser- Decay of HIV DNA in the Reservoir voir. In adults, TI has been associated with initially increased and the Impact of Short Treatment HIV DNA levels, which return to pre-TI levels aer >6  ft months Interruption in Kenyan Infants of ART resumption, suggesting that TI may not cause a lasting 1,2 3 2 increase in HIV-infected cell reservoirs [6, 9]. In contrast, a pre- Mark D. Pankau, Dalton Wamalwa, Sarah Benki-Nugent, 2 3 3 3 Kenneth Tapia, Evelyn Ngugi, Agnes Langat, Vincent Otieno, liminary report from the Children with HIV Early Antiretroviral 3 3 1,4 Helen Moraa, Elizabeth Maleche-Obimbo, Julie Overbaugh, era Th py trial (CHER) showed sustained increased HIV DNA 2,5,6,7 1,2 Grace C. John-Stewart, and Dara A. Lehman levels in 17 infants 2–3 years following a median 11-month TI 1 4 Division of Human Biology and Division of Public Health Sciences, Fred Hutchinson 2 5 Cancer Research Center, Seattle, Washington; Department of Global Health, Department [10], and a smaller study showed similar results [11]. To under- 6 7 of Medicine, Department of Pediatrics, and Department of Epidemiology, University of stand the impact of shorter TI in infants, we quantified blood Washington, Seattle, Washington; Department of Paediatrics, University of Nairobi, Nairobi, HIV DNA reservoir levels of Kenyan infants who were rand- Kenya omized to continued ART vs TI in the Optimizing Pediatric We compared change in HIV reservoir DNA following contin- HIV-1 Treatment study (OPH). ued antiretroviral therapy (ART) vs short treatment interrup- METHODS tion (TI) in early ART-treated Kenyan infants. While HIV DNA in the reservoir decayed with continued ART, HIV DNA levels Study Population were similar to pre-TI HIV DNA reservoir levels in most chil- OPH (NCT00428116) was a randomized controlled trial in dren aer s ft hort TI. which, following 24  months of continuous ART (median age Keywords. antiretroviral therapy; HIV DNA; infants; res- at initiation, 5  months), children were randomized to contin- ervoir; treatment interruption. ued ART or TI [12]. Blood was collected at 3-month intervals for CD4 measurements in real time and stored for HIV viral load (performed retrospectively). ART restart criteria were During acute HIV infection, a reservoir of long-lived infected CD4% <20–25%, or >one-third decrease from peak CD4, more cells is established that persists during antiretroviral treatment advanced World Health Organization stage, or weight-for-age (ART) and causes viral rebound upon ART cessation [1, 2]. decrease, as previously described [12]. After ART restart, there Experimental nonhuman primate models show that this reser- was no further interruption. Children were excluded from voir is generated within days of infection [3]. Initiating ART this substudy if HIV RNA was ≥1000 copies/mL during the early in HIV infection may limit reservoir size, increase time to 6  months prior to the HIV DNA measurements at 24  months viral rebound upon treatment cessation, and increase likelihood post–ART initiation (time of randomization) and 42  months of post-treatment viral control—but does not ensure remis- post–ART initiation (18  months following randomization; sion [2, 4–6]. In case reports, HIV-infected infants with early n = 27), or if samples were not available (n = 1). Seven of these ART had long periods of remission [7, 8], encouraging efforts children had virally suppressed samples available for HIV DNA to identify interventions that augment early ART to promote measurement at 75  months post–ART initiation (51  months post-treatment control in pediatric populations. Evaluating following randomization). these approaches requires analytical treatment interruption Laboratory Methods (TI). Thus, it is important to understand whether short TI Plasma HIV RNA was quantified using the Gen-Probe HIV-1 leads to sustained increases in latently infected cells in the HIV RNA assay (Gen Probe, San Diego, CA) with a limit of detec- reservoir. tion (LOD) of 2.18 log copies/mL. DNA was extracted from peripheral blood mononuclear cells (PBMCs) using QIAamp Received 6 July 2017; editorial decision 6 December 2017; accepted 8 December 2017. DNA (Qiagen, Valencia, CA). Cellular DNA was quantified Correspondence: D. A. Lehman, PhD, MHS, 1100 Fairview Ave N, C2-023, Seattle WA, 98109 using RPP30 ddPCR assay (Bio-Rad, Hercules, CA). HIV DNA (dlehman@fredhutch.org). Open Forum Infectious Diseases was quantified in duplicate by in-house cross-subtype pol pol- © The Author(s) 2017. Published by Oxford University Press on behalf of Infectious Diseases ymerase chain reaction (PCR) [13] modified for ddPCR, with Society of America. This is an Open Access article distributed under the terms of the Creative LOD of 5 copies/10 cells determined using previously validated Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any DNA from ACH2 cells that have a single HIV provirus per cell medium, provided the original work is not altered or transformed in any way, and that the work and HIV-negative genomic DNA controls (Supplementary is properly cited. For commercial re-use, please contact journals.permissions@oup.com DOI: 10.1093/ofid/ofx268 Figure  1). If results were below the LOD or were >2-fold BRIEF REPORT • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx268/4718123 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 6 discordant between duplicates, additional replicates were per- DNA copies/10 PBMCs (IQR, 24–174 HIV DNA copies/10 formed until >2.5e5 cells were tested. HIV DNA was normal- PBMCs) and 185 HIV DNA copies/10 PBMCs (IQR, 92–280 6 6 ized to RPP30 and HIV DNA copies/10 PBMCs reported. HIV DNA copies/10 PBMCs) in the continued and TI arms, respectively (P = .53). During the 18 months after randomiza- Statistical Analysis tion, 1 of 7 children in the continued arm had increased HIV Analysis was performed using R (version 3.4). HIV DNA fold DNA, 5 of 7 had decreased HIV DNA (range, 0.13–0.63), and 1 change was compared between continued and TI arms using of 7 children had HIV DNA levels below detection at both time the Wilcoxon rank-sum test. Correlation between postrandom- points (Figure 1B, Supplementary Table 1). In the TI arm, 3 of ization peak HIV RNA and HIV DNA fold change was deter- 7 children had increases in HIV DNA (range, 1.3–4.7), while 4 mined using Spearman’s rank-order correlation. of 7 had decreased or unchanged HIV DNA levels (range, 0.26– RESULTS 1.04). The median HIV DNA declines during the 18  months after randomization were –5.69 HIV DNA copies/1e6 PBMCs/ Cohort Characteristics During Initial 24 Months of ART month in the continued arm and 0.18 in the interrupted arm. During the OPH study, children were treated with 24  months The median HIV DNA fold changes were 0.32 (IQR, 0.22–0.58) of ART (range, 23–28  months), after which 42 were rand- in children randomized to continued ART and 1.04 (IQR, omized to TI (n  =  21) or continued ART (n  =  21). Fourteen 0.72–1.64) in children randomized to TI (P = .14) (Figure 1C). children from OPH met criteria for this laboratory substudy Similar results were observed when we excluded the 2 children (see “Methods”), 7 in each arm. At ART initiation, the median in the continued arm with postrandomization viremia: median age of the 14 infants was 4.8 months (interquartile range [IQR], HIV DNA fold changes were 0.32 (IQR, 0.23–0.46) vs 1.04 (IQR, 4.4–7 months), median CD4% was 22.5% (IQR, 15%–25%), and 0.71–1.64) in the continued and TI arms, respectively (P = .04). the median viral load was 6.5 log copies/mL (IQR, 5.7–6.9 log 10 10 In children with viremia during the 18 months after randomi- copies/mL) (Supplementary Table 1). Initial ART was NNRTI- zation, change in HIV DNA did not correlate with peak viremia based for 10 infants and PI-based for 4 infants. Twenty-four (Spearman’s rho, 0.12; P = .77). months following ART initiation (the time of randomization), In a limited number of children with longer follow-up (2 in 2 children were still on NNRTI-based ART, and 12 were tak- the TI arm and 5 in the continued arm), a virally suppressed ing PI-based ART; the median CD4% had risen to 35% (IQR, sample was available from 51  months aer ra ft ndomization 32%–40%), while the median viral load dropped below detec- (75 months aer ini ft tial ART). The median HIV DNA declines tion (Supplementary Table 1). from 0 to 51  months following randomization were –1.96 Treatment Interruption HIV DNA copies/1e6 PBMCs/month in the continued arm At the first postrandomization visit (~3 months after TI), all 7 and –0.37 in the interrupted arm. The median HIV DNA fold children in the TI arm exhibited viral rebound (Figure 1A) and changes were 0.44 (IQR, 0.44–0.75) and 0.92 (IQR, 0.84–1.00) met CD4-based criteria to restart ART, resulting in a median in children randomized to continued ART and TI, respectively of 106 days (IQR, 104–119 days) off ART. Of the 7 children in (Supplementary Figure 2). the continued arm, 5 had continuous viral suppression while 2 DISCUSSION had high HIV RNA levels at a single time point: infant #19 at 3 months and #1 at 9 months postrandomization (Figure  1A). We measured HIV DNA in the reservoir in children that Three months after randomization, the median viral load was started ART during the first year of life and were randomized 6.3 log copies/mL and was below detection (<2.18 log cop- 10 10 2 years later to continue or interrupt ART. We compared HIV ies/mL), and the median CD4% were 21% and 42% in the TI DNA levels at randomization and again 18 months later, after and continued arms, respectively. Eighteen months after ran- all children had resumed ART for >15  months and achieved domization, all 14 children were on PI-based ART with viral viral suppression. HIV DNA decayed in children with contin- loads below detection. The median CD4% were 34% (IQR, ued viral suppression, while the median HIV DNA fold change 30.5%–35.5%) in the TI group and 40% (IQR, 32.5%–42%) in after TI was 1.04, suggesting that TI lessens the decay that the continued group. occurs on continued ART. Indeed, for 2 children in the TI arm with ~4 years of follow-up, HIV DNA reservoir size remained HIV DNA Reservoir Dynamics relatively unchanged over time (Supplementary Figure 2). The After 24  months of initial ART, children randomized to con- fact that most children in the TI arm had similar HIV DNA tinued ART had a median of 168 HIV DNA copies/10 PBMCs levels before and after TI suggests that the reservoir reseed- (IQR, 101–235 HIV DNA copies/10 PBMCs) in the reservoir, ing that occurred during TI was followed by decay after ART and children in the TI arm had a median of 90 HIV DNA cop- 6 6 resumption. Mechanisms of reservoir seeding include new ies/10 PBMCs (IQR, 54–485 DNA copies/10 PBMCs; P = .9). infections of cells that become quiescent and clonal prolifera- Eighteen months later, after children in the TI arm had resumed tion of cells containing provirus [14, 15]. Our study could not ART for >15  months, HIV DNA levels were median 50 HIV 2 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx268/4718123 by Ed 'DeepDyve' Gillespie user on 16 March 2018 A Continued Interrupted 7 19 −30 −20 −10 01020 −30 −20 −10 01020 141 Months since randomization to continued or interrupted ART Continued Interrupted 0 18 (Before TI) (After TI) Months since randomization to continued or interrupted ART 10.0 1.0 0.1 Continued Interrupted Randomization Arm Figure 1. (A) Plasma HIV RNA copies/mL over time in infants randomized to continued antiretroviral therapy (ART; left) or interrupted ART (right). Limit of detection was 2.18 log10 copies HIV RNA/mL. (B) Comparison of total HIV DNA copies/10 peripheral blood mononuclear cells (PBMCs) before (24 month after initial ART) and after random- ization to treatment interruption (18 months later) in children in the continued arm (left) or interrupted arm (right). Limit of detection was 5 copies of HIV DNA/10 PBMCs. (C) HIV DNA fold change following randomization to treatment interruption (comparing HIV DNA at randomization and 18 months later) in infants randomized to continued (left) or interrupted ART (right). distinguish between these mechanisms as we did not charac- However, our data add substantially to the 2 previous studies terize the reservoir composition or replication competence on changes in HIV DNA following TI in children [10, 11], due to limited sample volume and cell viability. In addition, and they support results from adult cohorts [6, 9]. Analysis our cohort did not include infants treated with very early ART of 15 adults in the SPARTAC trial with transient TI showed (within hours of birth), which may have different viral reser- that HIV DNA returned to pre-TI levels aer ft ≥6  months of voir decay dynamics. ART [6]. Another TI study of 10 adults with very low HIV Our study did not quantify HIV DNA in tissue reservoirs and DNA levels in the reservoir prior to a median TI of 4 weeks was limited by small sample size with few evaluated time points. observed similar results, with HIV DNA levels returning to BRIEF REPORT • OFID • 3 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx268/4718123 by Ed 'DeepDyve' Gillespie user on 16 March 2018 HIV DNA fold change log HIV RNA copies/mL HIV DNA copies/10 PBMCs from month 24 to 42 Potential Conflicts of Interest. Conflicts that the editors consider relevant to pre-TI values aer t ft reatment resumption [9]. These studies the content of the manuscript have been disclosed. suggest that increases in HIV DNA can be minimized or reversed by rapid treatment resumption; however, larger studies References with longer follow-up are needed. 1. Chun TW, Engel D, Berrey MM, et  al. Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection. Proc Natl Acad Our findings add a new perspective that complements Sci U S A 1998; 95:8869–73. prior TI studies in perinatally infected infants, which focused 2. Persaud D, Palumbo PE, Ziemniak C, et al. Dynamics of the resting CD4(+) T-cell on longer TI. The largest pediatric TI reservoir study to date latent HIV reservoir in infants initiating HAART less than 6 months of age. AIDS 2012; 26:1483–90. included 17 infants treated earlier (<12 weeks at ART initia- 3. Whitney JB, Hill AL, Sanisetty S, et al. Rapid seeding of the viral reservoir prior to tion) and with longer TI (median, 11  months) than in our SIV viraemia in rhesus monkeys. Nature 2014; 512:74–7. 4. Li JZ, Etemad B, Ahmed H, et al. The size of the expressed HIV reservoir predicts study, and observed increased HIV DNA in the reservoir timing of viral rebound after treatment interruption. AIDS 2016; 30:343–53. 26  months aer t ft reatment resumption [10]. Another study 5. Sáez-Cirión A, Bacchus C, Hocqueloux L, et al; ANRS VISCONTI Study Group. Post-treatment HIV-1 controllers with a long-term virological remission after included only 3 infants with TI and showed 52.4-, 1.8-, and the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. 12.2-fold increases in HIV DNA following TI of 0.75, 6.8, and PLoS Pathog 2013; 9:e1003211. 6. Williams JP, Hurst J, Stöhr W, et  al; SPARTACTrial Investigators. HIV-1 DNA 71 months, respectively [11]. Here we show that a short inter- predicts disease progression and post-treatment virological control. Elife 2014; ruption of ~3 months does not appear to have sustained impact 3:e03821. on HIV DNA levels in the reservoir in children; however, it 7. Frange P, Faye A, Avettand-Fenoël V, et  al; ANRS EPF-CO10 Pediatric Cohort and the ANRS EP47 VISCONTI study group. HIV-1 virological remission lasting may lessen the rate of decay provided by early continued ART. more than 12 years after interruption of early antiretroviral therapy in a perina- These data suggest that reseeding of the reservoir in pediatric tally infected teenager enrolled in the French ANRS EPF-CO10 paediatric cohort: a case report. Lancet HIV 2016; 3:e49–54. HIV may be minimized with frequent viral load monitoring 8. Persaud D, Gay H, Ziemniak C, et al. Absence of detectable HIV-1 viremia after during short analytical TI. treatment cessation in an infant. N Engl J Med 2013; 369:1828–35. 9. Calin R, Hamimi C, Lambert-Niclot S, et  al; ULTRASTOP Study Group. Treatment interruption in chronically HIV-infected patients with an ultralow Supplementary Data HIV reservoir. AIDS 2016; 30:761–9. Supplementary materials are available at Open Forum Infectious Diseases 10. Payne H, Watters S, Hsaio M, et al. Early ART and sustained virologic suppression online. Consisting of data provided by the authors to benefit the reader, limits HIV proviral DNA reservoir: CHER evidence. In special issue: absracts the posted materials are not copyedited and are the sole responsibility of from the 2015 Conference on Retroviruses and Opportunistic Infections. Top the authors, so questions or comments should be addressed to the corre- Antiviral Med 2015; 23:16. sponding author. 11. Martínez-Bonet M, Puertas MC, Fortuny C, et al. Establishment and replenish- ment of the viral reservoir in perinatally HIV-1-infected children initiating very early antiretroviral therapy. Clin Infect Dis 2015; 61:1169–78. Acknowledgments 12. Wamalwa D, Benki-Nugent S, Langat A, et al. Treatment interruption after 2-year We thank the OPH study participants and their caregivers, without antiretroviral treatment initiated during acute/early HIV in infancy. AIDS 2016; whom this research would not be possible; and the OPH administrative, 30:2303–13. 13. Benki S, McClelland RS, Emery S, et al. Quantification of genital human immu- clinical, and data teams for their dedication and support. nodeficiency virus type 1 (HIV-1) DNA in specimens from women with low Financial support. This work was supported by the National Institute plasma HIV-1 RNA levels typical of HIV-1 nontransmitters. J Clin Microbiol of Allergy and Infectious Diseases (grant number R01 AI076105), the 2006; 44:4357–62. National Institute of Child Health and Development (grant numbers R01 14. Maldarelli F, Wu X, Su L, et  al. HIV latency. Specific HIV integration sites are HD023412, K24 HD054314  and R01 HD094718), and the University of linked to clonal expansion and persistence of infected cells. Science 2014; Washington Center for AIDS Research grant from the National Institutes 345:179–83. of Health([AI027757). 15. Wagner TA, McLaughlin S, Garg K, et al. HIV latency. Proliferation of cells with Potential conifl cts of interest. All authors: no reported conflicts of HIV integrated into cancer genes contributes to persistent infection. Science interest. All authors have submitted the ICMJE Form for Disclosure of 2014; 345:570–3. 4 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx268/4718123 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press
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Abstract

Open Forum Infectious Diseases BRIEF REPORT Few studies have measured the impact of TI on HIV reser- Decay of HIV DNA in the Reservoir voir. In adults, TI has been associated with initially increased and the Impact of Short Treatment HIV DNA levels, which return to pre-TI levels aer >6  ft months Interruption in Kenyan Infants of ART resumption, suggesting that TI may not cause a lasting 1,2 3 2 increase in HIV-infected cell reservoirs [6, 9]. In contrast, a pre- Mark D. Pankau, Dalton Wamalwa, Sarah Benki-Nugent, 2 3 3 3 Kenneth Tapia, Evelyn Ngugi, Agnes Langat, Vincent Otieno, liminary report from the Children with HIV Early Antiretroviral 3 3 1,4 Helen Moraa, Elizabeth Maleche-Obimbo, Julie Overbaugh, era Th py trial (CHER) showed sustained increased HIV DNA 2,5,6,7 1,2 Grace C. John-Stewart, and Dara A. Lehman levels in 17 infants 2–3 years following a median 11-month TI 1 4 Division of Human Biology and Division of Public Health Sciences, Fred Hutchinson 2 5 Cancer Research Center, Seattle, Washington; Department of Global Health, Department [10], and a smaller study showed similar results [11]. To under- 6 7 of Medicine, Department of Pediatrics, and Department of Epidemiology, University of stand the impact of shorter TI in infants, we quantified blood Washington, Seattle, Washington; Department of Paediatrics, University of Nairobi, Nairobi, HIV DNA reservoir levels of Kenyan infants who were rand- Kenya omized to continued ART vs TI in the Optimizing Pediatric We compared change in HIV reservoir DNA following contin- HIV-1 Treatment study (OPH). ued antiretroviral therapy (ART) vs short treatment interrup- METHODS tion (TI) in early ART-treated Kenyan infants. While HIV DNA in the reservoir decayed with continued ART, HIV DNA levels Study Population were similar to pre-TI HIV DNA reservoir levels in most chil- OPH (NCT00428116) was a randomized controlled trial in dren aer s ft hort TI. which, following 24  months of continuous ART (median age Keywords. antiretroviral therapy; HIV DNA; infants; res- at initiation, 5  months), children were randomized to contin- ervoir; treatment interruption. ued ART or TI [12]. Blood was collected at 3-month intervals for CD4 measurements in real time and stored for HIV viral load (performed retrospectively). ART restart criteria were During acute HIV infection, a reservoir of long-lived infected CD4% <20–25%, or >one-third decrease from peak CD4, more cells is established that persists during antiretroviral treatment advanced World Health Organization stage, or weight-for-age (ART) and causes viral rebound upon ART cessation [1, 2]. decrease, as previously described [12]. After ART restart, there Experimental nonhuman primate models show that this reser- was no further interruption. Children were excluded from voir is generated within days of infection [3]. Initiating ART this substudy if HIV RNA was ≥1000 copies/mL during the early in HIV infection may limit reservoir size, increase time to 6  months prior to the HIV DNA measurements at 24  months viral rebound upon treatment cessation, and increase likelihood post–ART initiation (time of randomization) and 42  months of post-treatment viral control—but does not ensure remis- post–ART initiation (18  months following randomization; sion [2, 4–6]. In case reports, HIV-infected infants with early n = 27), or if samples were not available (n = 1). Seven of these ART had long periods of remission [7, 8], encouraging efforts children had virally suppressed samples available for HIV DNA to identify interventions that augment early ART to promote measurement at 75  months post–ART initiation (51  months post-treatment control in pediatric populations. Evaluating following randomization). these approaches requires analytical treatment interruption Laboratory Methods (TI). Thus, it is important to understand whether short TI Plasma HIV RNA was quantified using the Gen-Probe HIV-1 leads to sustained increases in latently infected cells in the HIV RNA assay (Gen Probe, San Diego, CA) with a limit of detec- reservoir. tion (LOD) of 2.18 log copies/mL. DNA was extracted from peripheral blood mononuclear cells (PBMCs) using QIAamp Received 6 July 2017; editorial decision 6 December 2017; accepted 8 December 2017. DNA (Qiagen, Valencia, CA). Cellular DNA was quantified Correspondence: D. A. Lehman, PhD, MHS, 1100 Fairview Ave N, C2-023, Seattle WA, 98109 using RPP30 ddPCR assay (Bio-Rad, Hercules, CA). HIV DNA (dlehman@fredhutch.org). Open Forum Infectious Diseases was quantified in duplicate by in-house cross-subtype pol pol- © The Author(s) 2017. Published by Oxford University Press on behalf of Infectious Diseases ymerase chain reaction (PCR) [13] modified for ddPCR, with Society of America. This is an Open Access article distributed under the terms of the Creative LOD of 5 copies/10 cells determined using previously validated Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any DNA from ACH2 cells that have a single HIV provirus per cell medium, provided the original work is not altered or transformed in any way, and that the work and HIV-negative genomic DNA controls (Supplementary is properly cited. For commercial re-use, please contact journals.permissions@oup.com DOI: 10.1093/ofid/ofx268 Figure  1). If results were below the LOD or were >2-fold BRIEF REPORT • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx268/4718123 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 6 discordant between duplicates, additional replicates were per- DNA copies/10 PBMCs (IQR, 24–174 HIV DNA copies/10 formed until >2.5e5 cells were tested. HIV DNA was normal- PBMCs) and 185 HIV DNA copies/10 PBMCs (IQR, 92–280 6 6 ized to RPP30 and HIV DNA copies/10 PBMCs reported. HIV DNA copies/10 PBMCs) in the continued and TI arms, respectively (P = .53). During the 18 months after randomiza- Statistical Analysis tion, 1 of 7 children in the continued arm had increased HIV Analysis was performed using R (version 3.4). HIV DNA fold DNA, 5 of 7 had decreased HIV DNA (range, 0.13–0.63), and 1 change was compared between continued and TI arms using of 7 children had HIV DNA levels below detection at both time the Wilcoxon rank-sum test. Correlation between postrandom- points (Figure 1B, Supplementary Table 1). In the TI arm, 3 of ization peak HIV RNA and HIV DNA fold change was deter- 7 children had increases in HIV DNA (range, 1.3–4.7), while 4 mined using Spearman’s rank-order correlation. of 7 had decreased or unchanged HIV DNA levels (range, 0.26– RESULTS 1.04). The median HIV DNA declines during the 18  months after randomization were –5.69 HIV DNA copies/1e6 PBMCs/ Cohort Characteristics During Initial 24 Months of ART month in the continued arm and 0.18 in the interrupted arm. During the OPH study, children were treated with 24  months The median HIV DNA fold changes were 0.32 (IQR, 0.22–0.58) of ART (range, 23–28  months), after which 42 were rand- in children randomized to continued ART and 1.04 (IQR, omized to TI (n  =  21) or continued ART (n  =  21). Fourteen 0.72–1.64) in children randomized to TI (P = .14) (Figure 1C). children from OPH met criteria for this laboratory substudy Similar results were observed when we excluded the 2 children (see “Methods”), 7 in each arm. At ART initiation, the median in the continued arm with postrandomization viremia: median age of the 14 infants was 4.8 months (interquartile range [IQR], HIV DNA fold changes were 0.32 (IQR, 0.23–0.46) vs 1.04 (IQR, 4.4–7 months), median CD4% was 22.5% (IQR, 15%–25%), and 0.71–1.64) in the continued and TI arms, respectively (P = .04). the median viral load was 6.5 log copies/mL (IQR, 5.7–6.9 log 10 10 In children with viremia during the 18 months after randomi- copies/mL) (Supplementary Table 1). Initial ART was NNRTI- zation, change in HIV DNA did not correlate with peak viremia based for 10 infants and PI-based for 4 infants. Twenty-four (Spearman’s rho, 0.12; P = .77). months following ART initiation (the time of randomization), In a limited number of children with longer follow-up (2 in 2 children were still on NNRTI-based ART, and 12 were tak- the TI arm and 5 in the continued arm), a virally suppressed ing PI-based ART; the median CD4% had risen to 35% (IQR, sample was available from 51  months aer ra ft ndomization 32%–40%), while the median viral load dropped below detec- (75 months aer ini ft tial ART). The median HIV DNA declines tion (Supplementary Table 1). from 0 to 51  months following randomization were –1.96 Treatment Interruption HIV DNA copies/1e6 PBMCs/month in the continued arm At the first postrandomization visit (~3 months after TI), all 7 and –0.37 in the interrupted arm. The median HIV DNA fold children in the TI arm exhibited viral rebound (Figure 1A) and changes were 0.44 (IQR, 0.44–0.75) and 0.92 (IQR, 0.84–1.00) met CD4-based criteria to restart ART, resulting in a median in children randomized to continued ART and TI, respectively of 106 days (IQR, 104–119 days) off ART. Of the 7 children in (Supplementary Figure 2). the continued arm, 5 had continuous viral suppression while 2 DISCUSSION had high HIV RNA levels at a single time point: infant #19 at 3 months and #1 at 9 months postrandomization (Figure  1A). We measured HIV DNA in the reservoir in children that Three months after randomization, the median viral load was started ART during the first year of life and were randomized 6.3 log copies/mL and was below detection (<2.18 log cop- 10 10 2 years later to continue or interrupt ART. We compared HIV ies/mL), and the median CD4% were 21% and 42% in the TI DNA levels at randomization and again 18 months later, after and continued arms, respectively. Eighteen months after ran- all children had resumed ART for >15  months and achieved domization, all 14 children were on PI-based ART with viral viral suppression. HIV DNA decayed in children with contin- loads below detection. The median CD4% were 34% (IQR, ued viral suppression, while the median HIV DNA fold change 30.5%–35.5%) in the TI group and 40% (IQR, 32.5%–42%) in after TI was 1.04, suggesting that TI lessens the decay that the continued group. occurs on continued ART. Indeed, for 2 children in the TI arm with ~4 years of follow-up, HIV DNA reservoir size remained HIV DNA Reservoir Dynamics relatively unchanged over time (Supplementary Figure 2). The After 24  months of initial ART, children randomized to con- fact that most children in the TI arm had similar HIV DNA tinued ART had a median of 168 HIV DNA copies/10 PBMCs levels before and after TI suggests that the reservoir reseed- (IQR, 101–235 HIV DNA copies/10 PBMCs) in the reservoir, ing that occurred during TI was followed by decay after ART and children in the TI arm had a median of 90 HIV DNA cop- 6 6 resumption. Mechanisms of reservoir seeding include new ies/10 PBMCs (IQR, 54–485 DNA copies/10 PBMCs; P = .9). infections of cells that become quiescent and clonal prolifera- Eighteen months later, after children in the TI arm had resumed tion of cells containing provirus [14, 15]. Our study could not ART for >15  months, HIV DNA levels were median 50 HIV 2 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx268/4718123 by Ed 'DeepDyve' Gillespie user on 16 March 2018 A Continued Interrupted 7 19 −30 −20 −10 01020 −30 −20 −10 01020 141 Months since randomization to continued or interrupted ART Continued Interrupted 0 18 (Before TI) (After TI) Months since randomization to continued or interrupted ART 10.0 1.0 0.1 Continued Interrupted Randomization Arm Figure 1. (A) Plasma HIV RNA copies/mL over time in infants randomized to continued antiretroviral therapy (ART; left) or interrupted ART (right). Limit of detection was 2.18 log10 copies HIV RNA/mL. (B) Comparison of total HIV DNA copies/10 peripheral blood mononuclear cells (PBMCs) before (24 month after initial ART) and after random- ization to treatment interruption (18 months later) in children in the continued arm (left) or interrupted arm (right). Limit of detection was 5 copies of HIV DNA/10 PBMCs. (C) HIV DNA fold change following randomization to treatment interruption (comparing HIV DNA at randomization and 18 months later) in infants randomized to continued (left) or interrupted ART (right). distinguish between these mechanisms as we did not charac- However, our data add substantially to the 2 previous studies terize the reservoir composition or replication competence on changes in HIV DNA following TI in children [10, 11], due to limited sample volume and cell viability. In addition, and they support results from adult cohorts [6, 9]. Analysis our cohort did not include infants treated with very early ART of 15 adults in the SPARTAC trial with transient TI showed (within hours of birth), which may have different viral reser- that HIV DNA returned to pre-TI levels aer ft ≥6  months of voir decay dynamics. ART [6]. Another TI study of 10 adults with very low HIV Our study did not quantify HIV DNA in tissue reservoirs and DNA levels in the reservoir prior to a median TI of 4 weeks was limited by small sample size with few evaluated time points. observed similar results, with HIV DNA levels returning to BRIEF REPORT • OFID • 3 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx268/4718123 by Ed 'DeepDyve' Gillespie user on 16 March 2018 HIV DNA fold change log HIV RNA copies/mL HIV DNA copies/10 PBMCs from month 24 to 42 Potential Conflicts of Interest. Conflicts that the editors consider relevant to pre-TI values aer t ft reatment resumption [9]. These studies the content of the manuscript have been disclosed. suggest that increases in HIV DNA can be minimized or reversed by rapid treatment resumption; however, larger studies References with longer follow-up are needed. 1. Chun TW, Engel D, Berrey MM, et  al. Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection. Proc Natl Acad Our findings add a new perspective that complements Sci U S A 1998; 95:8869–73. prior TI studies in perinatally infected infants, which focused 2. Persaud D, Palumbo PE, Ziemniak C, et al. Dynamics of the resting CD4(+) T-cell on longer TI. The largest pediatric TI reservoir study to date latent HIV reservoir in infants initiating HAART less than 6 months of age. AIDS 2012; 26:1483–90. included 17 infants treated earlier (<12 weeks at ART initia- 3. Whitney JB, Hill AL, Sanisetty S, et al. Rapid seeding of the viral reservoir prior to tion) and with longer TI (median, 11  months) than in our SIV viraemia in rhesus monkeys. Nature 2014; 512:74–7. 4. Li JZ, Etemad B, Ahmed H, et al. The size of the expressed HIV reservoir predicts study, and observed increased HIV DNA in the reservoir timing of viral rebound after treatment interruption. AIDS 2016; 30:343–53. 26  months aer t ft reatment resumption [10]. Another study 5. Sáez-Cirión A, Bacchus C, Hocqueloux L, et al; ANRS VISCONTI Study Group. Post-treatment HIV-1 controllers with a long-term virological remission after included only 3 infants with TI and showed 52.4-, 1.8-, and the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. 12.2-fold increases in HIV DNA following TI of 0.75, 6.8, and PLoS Pathog 2013; 9:e1003211. 6. Williams JP, Hurst J, Stöhr W, et  al; SPARTACTrial Investigators. HIV-1 DNA 71 months, respectively [11]. Here we show that a short inter- predicts disease progression and post-treatment virological control. Elife 2014; ruption of ~3 months does not appear to have sustained impact 3:e03821. on HIV DNA levels in the reservoir in children; however, it 7. Frange P, Faye A, Avettand-Fenoël V, et  al; ANRS EPF-CO10 Pediatric Cohort and the ANRS EP47 VISCONTI study group. HIV-1 virological remission lasting may lessen the rate of decay provided by early continued ART. more than 12 years after interruption of early antiretroviral therapy in a perina- These data suggest that reseeding of the reservoir in pediatric tally infected teenager enrolled in the French ANRS EPF-CO10 paediatric cohort: a case report. Lancet HIV 2016; 3:e49–54. HIV may be minimized with frequent viral load monitoring 8. Persaud D, Gay H, Ziemniak C, et al. Absence of detectable HIV-1 viremia after during short analytical TI. treatment cessation in an infant. N Engl J Med 2013; 369:1828–35. 9. Calin R, Hamimi C, Lambert-Niclot S, et  al; ULTRASTOP Study Group. Treatment interruption in chronically HIV-infected patients with an ultralow Supplementary Data HIV reservoir. AIDS 2016; 30:761–9. Supplementary materials are available at Open Forum Infectious Diseases 10. Payne H, Watters S, Hsaio M, et al. Early ART and sustained virologic suppression online. Consisting of data provided by the authors to benefit the reader, limits HIV proviral DNA reservoir: CHER evidence. In special issue: absracts the posted materials are not copyedited and are the sole responsibility of from the 2015 Conference on Retroviruses and Opportunistic Infections. Top the authors, so questions or comments should be addressed to the corre- Antiviral Med 2015; 23:16. sponding author. 11. Martínez-Bonet M, Puertas MC, Fortuny C, et al. Establishment and replenish- ment of the viral reservoir in perinatally HIV-1-infected children initiating very early antiretroviral therapy. Clin Infect Dis 2015; 61:1169–78. Acknowledgments 12. Wamalwa D, Benki-Nugent S, Langat A, et al. Treatment interruption after 2-year We thank the OPH study participants and their caregivers, without antiretroviral treatment initiated during acute/early HIV in infancy. AIDS 2016; whom this research would not be possible; and the OPH administrative, 30:2303–13. 13. Benki S, McClelland RS, Emery S, et al. Quantification of genital human immu- clinical, and data teams for their dedication and support. nodeficiency virus type 1 (HIV-1) DNA in specimens from women with low Financial support. This work was supported by the National Institute plasma HIV-1 RNA levels typical of HIV-1 nontransmitters. J Clin Microbiol of Allergy and Infectious Diseases (grant number R01 AI076105), the 2006; 44:4357–62. National Institute of Child Health and Development (grant numbers R01 14. Maldarelli F, Wu X, Su L, et  al. HIV latency. Specific HIV integration sites are HD023412, K24 HD054314  and R01 HD094718), and the University of linked to clonal expansion and persistence of infected cells. Science 2014; Washington Center for AIDS Research grant from the National Institutes 345:179–83. of Health([AI027757). 15. Wagner TA, McLaughlin S, Garg K, et al. HIV latency. Proliferation of cells with Potential conifl cts of interest. All authors: no reported conflicts of HIV integrated into cancer genes contributes to persistent infection. Science interest. All authors have submitted the ICMJE Form for Disclosure of 2014; 345:570–3. 4 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx268/4718123 by Ed 'DeepDyve' Gillespie user on 16 March 2018

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Open Forum Infectious DiseasesOxford University Press

Published: Jan 1, 2018

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