Abstract
Importance
Combination antiretroviral therapy (cART) initiated within several weeks of HIV infection in adults limits proviral reservoirs that preclude HIV cure. Biomarkers of restricted proviral reservoirs may aid in the monitoring of HIV remission or cure.
Objectives
To quantify peripheral blood proviral reservoir size in perinatally HIV-infected adolescents and to identify correlates of limited proviral reservoirs.
Design, Setting, and Participants
A cross-sectional study including 144 perinatally HIV-infected (PHIV+) youth (median age: 14.3 years), enrolled in the US-based Pediatric HIV/AIDS Cohort Study, on durable (median: 10.2 years) cART, stratified by age at virologic control.
Main Outcome and Measures
The primary endpoint was peripheral blood mononuclear cell (PBMC) proviral load following virologic control at different ages. Correlations between proviral load and markers of active HIV production (HIV-specific antibodies, 2-long terminal repeat (2-LTR) circles), and markers of immune activation and inflammation were also assessed.
Results
Proviral reservoir size was markedly reduced in the PHIV+ youth who achieved virologic control by age 1 year (4.2 [interquartile range, 2.6-8 6] copies per 1 million PBMCs) compared to those who achieved virologic control between 1-5 years of age (19.4 [interquartile range, 5.5-99.8] copies per 1 million PBMCs) or after age 5 years (−(70.7 [interquartile range, 23.2-209.4] copies per 1 million PBMCs; P < .00l). A proviral burden <10 copies/million PBMCs was measured in 11 (79%), 20 (40%), and 13 (18%) participants with virologic control at ages <1 year, 1-5 years, and >5 years, respectively (p<0.001). Lower proviral load was associated with undetectable 2-LTR circles (p<0.001) and HIV negative or indeterminate serostatus (p<0.001), but not with concentrations of soluble immune activation markers CD14 and CD163.
Conclusions and Relevance
Early effective cART along with prolonged virologic suppression after perinatal HIV infection leads to negligible peripheral blood proviral reservoirs in adolescence and is associated with negative or indeterminate HIV serostatus. These findings highlight the long-term effect of early effective control of HIV replication on biomarkers of HIV persistence in perinatal infection and the utility of HIV serostatus as a biomarker for small proviral reservoir size, though not necessarily of cure.
INTRODUCTION
Despite progress in the prevention of mother-to-child HIV transmission, nearly 260,000 infections occurred among children in 2012.1 Pediatric HIV infection remains a public health problem, but early treatment improves survival and preserves immune health.2-4 Cure of HIV infection remains elusive due to the early establishment of viral latency in long-lived resting memory CD4+ T cells that evade immune surveillance mechanisms and antiretroviral drugs.5 Sufficiently reducing replication-competent HIV reservoirs to achieve HIV cure or remission where discontinuation of combination antiretroviral therapy (cART) does not lead to viremic rebound is under intense study.6 cART during acute HIV infection limits HIV reservoir size 7-10, and leads to HIV remission in a subset of HIV-infected adults (post-treatment controllers).11;12 We recently reported a case of viral remission in a perinatally HIV-infected (PHIV+) child following 18 months of cART started at 30 hours of age.13
In post-treatment controllers, a mean proviral load of 116 copies/million peripheral blood mononuclear cells (PBMCs) before treatment discontinuation was associated with virologic control for a median of six years after cART discontinuation.12 Small proviral reservoir size is also reported in Elite Controllers in whom host immune mechanisms control HIV replication in the absence of cART.14 Knowing whether long-term virologic suppression with cART can durably reduce HIV reservoirs in perinatal infection is important for studying viral remission or cure. In this study, we examined the impact of age at virologic control after cART on the size of peripheral blood HIV reservoirs in perinatally-infected youth with long-term virologic control (≥10 years) and evaluated the association between reservoir size and measures of ongoing HIV replication or production such as 2-LTR circles, immune activation, and HIV-specific immune responses.
MATERIALS AND METHODS
Study Participants
The source population for this study was the Pediatric HIV/AIDS Cohort Study (PHACS) Adolescent Master Protocol (AMP), a prospective cohort study designed to evaluate the impact of HIV infection and antiretroviral treatment (ART) on perinatally-infected youth. PHACS/AMP enrolled 451 PHIV+ children and adolescents and 227 perinatally HIV-exposed uninfected (PHEU) youth receiving care at 15 sites in the United States.15 PHACS/AMP was approved by the institutional review board (IRB) at the Harvard School of Public Health and at each participating site. Written informed consent was obtained from each participant’s parent or legal guardian. Assent was obtained from child participants according to local IRB guidelines.
At each study visit, participant demographics, ART history, HIV viral loads (VLs), and CD4+ T cell counts were collected, along with repository specimens of plasma and cryopreserved PBMCs. ART was initiated based on contemporary treatment guidelines. Of 430 PHIV+ children and adolescents enrolled in PHACS/AMP who initiated cART, those who achieved and maintained virologic control as of their last VL measurement before July 2, 2012 were eligible for inclusion (Figure 1). Virologic control was defined as having two consecutive VLs <400 copies/milliliter (mL) of plasma following cART initiation and maintaining VLs <400 copies/mL but allowing for isolated VLs ≥400 copies/mL (i.e., intermittent viremia). Within the period of virologic control, the most recent pair (<30 days apart) of plasma and PBMC specimens was analyzed for proviral burden, immune activation and inflammation, and HIV serostatus. If PBMCs were not available, the most recent plasma specimen was used for testing of HIV serostatus and immune activation markers. The final study population included 144 PHIV+ youth with complete information on proviral load, immune activation and inflammation, and HIV serostatus (Figure 1). Ten randomly selected, age-matched PHEU adolescents enrolled in PHACS/AMP served as controls for analysis of immune activation markers.
Figure 1.
Derivation of study population.
Quantification of HIV DNA and 2-Long Terminal Repeat Circles
HIV and genomic DNA were extracted from frozen PBMC pellets using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) and measured in triplicate using ultrasensitive droplet digital PCR (ddPCR).16 Proviral burden was expressed as HIV DNA copies/million PBMCs. 2-long terminal repeat (2-LTR) circles were also quantified in the same reaction as used for proviral DNA quantification.16 With ddPCR, the RNase P gene is used to establish the cellular equivalent of genomic DNA analyzed per reaction.
Determination of Immune Activation
Immune activation was assessed by measuring levels of soluble CD14 (sCD14) and soluble CD163 (sCD163) in stored plasma using the Quantikine Human sCD14 and soluble CD163 Immunoassays (R&D Systems, Minneapolis, MN). Samples were tested in duplicate. The optical density was determined using Softmax VersaMaxPLUS ROM v1.21 reader. The concentrations of sCD14 and sCD163 were determined by interpolation to a nonlinear standard curve of sCD14 dilutions and sCD163 using GraphPad Prism v5. The Meso Scale Discovery Pro-inflammatory 9-plex assay (Rockville, MD) was used to measure, in duplicate, the following pro-inflammatory cytokines: IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, GM-CSF, IFN-γ, and TNF-α.
HIV-Specific Antibody Detection
HIV antibody testing was performed using enzyme immunosoassay (GS HIV-1/2 PLUS O EIA, Biorad, Hercules, CA) per the manufacturer’s instruction. Western blot testing assessed antibody responses to gp160, gp120, p66, p55, p51, gp41, p31, p24, and p17 per the manufacturer’s instructions (Cambridge Biotech HIV-1 Western Blot Kit, Maxim Biomedical, Inc. Rockville, MD) and was interpreted as seronegative if no bands were present; seropositive if any two or more of the following bands were present: p24, gp41 and gp120/160; and indeterminate if any bands were present but did not meet the criteria for seropositivity.
Statistical Analyses
The date of the PBMC specimen was considered to be the last visit or end of follow-up for analyses. Demographic and clinical characteristics were compared by age at virologic control after cART initiation (<1 year, 1-5 years, >5 years) using Fisher’s Exact tests and Kruskal-Wallis tests as appropriate. Proviral load, 2-LTR circles, and HIV serostatus were also compared by age at virologic control using Fisher’s Exact and Kruskal-Wallis tests. Distributions of proviral loads among those with and without detectable 2-LTR circles and with positive or negative/indeterminate HIV serostatus were compared using the Kruskal-Wallis test. Values of proviral load below the limit of detection (LOD) were set to the LOD when examining the distribution of proviral load. Immune activation and inflammation markers were compared by age at virologic control and to the PHEU comparison group as continuous parameters. Their distributions were also compared by 2-LTR circle detection. The Spearman’s rank correlation coefficient was used to assess the correlation between each immune activation and inflammation marker and proviral load. All analyses were conducted using SAS version 9.2 (SAS Institute, Cary NC).
RESULTS
Study Participant Characteristics
The 144 PHIV+ study participants were mostly non-Hispanic blacks (66%) and 49% were female (Table 1). The median age at cART initiation was 2.4 months for the 14 youth achieving virologic control by age one year, 1.9 years for the 53 adolescents who achieved control between ages 1 and 5, and 5.6 years for the 77 who achieved virologic control after age five years (Table 1). Those who achieved virologic control after five years of age were more likely to have received non-cART regimens before cART initiation and took longer to achieve virologic control after cART initiation (Table 1). Higher plasma VL concentrations (p=0.01) and CD4+ T cell percentage at cART initiation (p=0.07) were associated with younger age at virologic control. Younger age at virologic control was also highly correlated with a longer duration of virologic control. The median duration of virologic control from confirmed virologic suppression to the end of follow-up was 11.8 years, 9.6 years and 4.4 years for youth achieving virologic control by age one year, by 1-5 years of age, and after 5 years of age, respectively (Table 1).
Table 1. Descriptive characteristics by age at virologic control.
| Age at virologic control | |||||
|---|---|---|---|---|---|
| Characteristics | Total (N=144) |
<1 year old (N=14) |
1 to 5 years old (N=53) |
>5 years old (N=77) |
P-Value |
| Sex | |||||
| M | 74 (51%) | 7 (50%) | 25 (47%) | 42 (55%) | 0.709† |
| F | 70 (49%) | 7 (50%) | 28 (53%) | 35 (45%) | |
| Year of birth | |||||
| Median (Q1, Q3) |
1,996 (1,994, 1,999) |
1,999 (1,997, 2,000) |
1,998 (1,995, 2,000) |
1,995 (1,993, 1,997) |
<.001‡ |
| Race/ethnicity | |||||
| White Non-Hispanic/Other | 17 (12%) | 2 (14%) | 6 (11%) | 9 (12%) | 0.066† |
| Black Non-Hispanic | 95 (66%) | 10 (71%) | 40 (75%) | 45 (58%) | |
| Hispanic (Regardless of Race) | 30 (21%) | 2 (14%) | 5 (9%) | 23 (30%) | |
| Missing | 2 (1%) | 0 (0%) | 2 (4%) | 0 (0%) | |
| Pre-cART ART regimens (#) | |||||
| Median (Q1, Q3) |
1 (0, 2) |
0 (0, 0) |
0 (0, 2) |
1 (0, 3) |
<.001‡ |
| Pre-cART ART regimens (years duration) | |||||
| Median (Q1, Q3) |
0.2 (0.0, 2.9) |
0.0 (0.0, 0.0) |
0.0 (0.0, 1.0) |
1.6 (0.0, 5.4) |
<.001‡ |
| First ever cART type | |||||
| PI alone | 93 (65%) | 10 (71%) | 38 (72%) | 45 (58%) | 0.621 † |
| PI + NNRTI | 19 (13%) | 1 (7%) | 6 (11%) | 12 (16%) | |
| NNRTI alone | 32 (22%) | 3 (21%) | 9 (17%) | 20 (26%) | |
| Age at cART initiation (years) | |||||
| Median (Q1, Q3) |
2.7 (0.8, 5.8) |
0.2 (0.1, 0.3) |
1.9 (0.8, 3.3) |
5.6 (2.1, 9.3) |
<.001‡ |
| HIV viral load at cART initiation (log10 copies/mL) |
|||||
| Median (Q1, Q3) |
4.9 (3.9, 5.4) |
5.6 (3.9, 6.3) |
5.0 (4.7, 5.4) |
4.5 (3.5, 5.2) |
0.007‡ |
| # missing | 24 | 0 | 8 | 16 | |
| CD4% at cART initiation | |||||
| Median (Q1, Q3) |
27 (20, 35) |
32 (27, 42) |
28 (21, 37) |
26 (19, 34) |
0.065‡ |
| # missing | 23 | 1 | 8 | 14 | |
| CDC class at cART initiation | |||||
| N or A | 97 (67%) | 12 (86%) | 36 (68%) | 49 (64%) | 0.180† |
| B | 20 (14%) | 2 (14%) | 9 (17%) | 9 (12%) | |
| C | 27 (19%) | 0 (0%) | 8 (15%) | 19 (25%) | |
| Age at virologic control (years) | |||||
| Median (Q1, Q3) |
6.7 (2.9, 10.6) |
0.7 (0.6, 0.9) |
3.4 (2.5, 4.8) |
10.2 (8.0, 12.1) |
<.001‡ |
| Years from cART initiation to virologic control | |||||
| Median (Q1, Q3) |
1.4 (0.4, 4.7) |
0.5 (0.3, 4.7) |
0.8 (0.4, 1.9) |
4.3 (0.6, 7.5) |
<.001‡ |
| Age at last visit (years)§ | |||||
| Median (Q1, Q3) |
14.3 (12.0, 16.5) |
12.6 (11.1, 14.0) |
12.7 (10.4, 15.4) |
15.8 (13.8, 17.2) |
<.001‡ |
| HIV viral load at last visit (log10 copies/mL)§ | |||||
| Median (Q1, Q3) |
1.7 (1.6, 1.7) |
1.7 (1.6, 1.7) |
1.7 (1.6, 1.7) |
1.7 (1.6, 1.7) |
0.299‡ |
| # missing | 2 | 0 | 0 | 2 | |
| CD4% at last visit§ | |||||
| Median (Q1, Q3) |
37 (34, 42) |
40 (37, 43) |
38 (36, 42) |
36 (33, 41) |
0.034‡ |
| # missing | 5 | 0 | 4 | 1 | |
| CD4 count at last visit§ | |||||
| Median (Q1, Q3) |
779 (621, 992) |
756 (553, 1,220) |
810 (673, 1,122) |
761 (619, 912) |
0.234‡ |
| # missing | 5 | 0 | 4 | 1 | |
| CD4/CD8 count ratio at last visit§ | |||||
| Median (Q1, Q3) |
1.3 (10, 1.6) |
1.4 (12, 1.7) |
1.5 (1.1, 1.8) |
1.2 (0.9, 1.5) |
0.004‡ |
| # missing | 7 | 0 | 5 | 2 | |
| Duration of cART use through last visit (years)§ |
|||||
| Median (Q1, Q3) |
10.2 (7.0, 12.7) |
12.4 (11.0, 13.7) |
10.9 (8.5, 12.7) |
8.3 (6.0, 12.0) |
<.001‡ |
| Duration of virologic control through last visit (years)§ |
|||||
| Median (Q1, Q3) |
7.2 (4.1, 10.5) |
11.8 (10.7, 13.4) |
9.6 (7.1, 12.1) |
4.4 (2.4, 7.0) |
<.001‡ |
= Fisher’s exact test.
= Kruskal-Wallis test.
= Last visit is defined by the date of the PBMC specimen analyzed.
At the end of follow-up, the median age of PHIV+ adolescents was 14.3 years (interquartile range [IQR]: 12.0, 16.5); adolescents who suppressed HIV after age 5 years were significantly older than those suppressing at younger ages (Table 1). CD4+ T cells reconstituted to slightly higher percentages in youth with virologic control at younger ages (p=0.03), but the increase in median CD4+ T cell percentages from cART initiation to the end of follow-up was similar between groups. Among the 10 PHEU adolescents used as a comparison group for the immune activation and inflammation markers, 40% were non-Hispanic blacks, 30% were female, and their median age at the time of plasma specimen collection was 13.9 years (IQR: 12.5, 15.4).
Peripheral Blood HIV Proviral Burden at the Last Visit
The median proviral load in PBMCs at the last visit was significantly lower in subjects achieving virologic control by age one year (4.2 copies/million PBMCs [IQR; 2.6, 8.6]) compared with those achieving virologic control between ages 1-5 and after age 5 years, in whom the median proviral loads were 19.4 copies/million PBMCs (IQR: 5.5, 99.8) and 70.7 copies/million PBMCs (IQR: 23.2, 209.4), respectively (p<0.001) (Figure 2). The ddPCR LOD varies based on the number of analyzed cells available in each sample. The median LOD for HIV DNA was 2.8 copies/million PBMCs (IQR: 1.9, 3.8) and 6 samples had a LOD of 10 copies/million PBMCs or higher due to limited cell numbers. A proviral burden <10 copies/million PBMCs was measured in 11(79%), 20(40%), and 13(18%) PHIV+ youth with virologic control at ages <1 year, 1-5 years, and >5 years, respectively (p<0.001). Forty-six percent of 13 who achieved virologic control by age one year had proviral DNA <4 copies/million PBMCs compared with 11% of 101 adolescents who achieved control after age one year (p=0.01).
Figure 2.
Distribution of proviral load at last visit by age at virologic control. The limit of detection for proviral load varies based on the number of cells available for analysis. Values of proviral load below the limit of detection were set to the limit of detection. Filled and open shapes represent samples tested above and below PBMC assay detection limit, respectively. Triangles and circles represent samples tested above and below 2-LTR circle assay detection limit, respectively.
Ongoing Viremia and Virus Replication during Long-term cART at the Last Visit
Intermittent viremia was rare. Of 3,997 plasma VL measurements collected over a median of 7.2 years from the time of virologic suppression to the end of follow-up, only 98 (2.5%) were ≥400 copies/mL and 11 (0.3%) were ≥10,000 copies/mL. Twenty-nine (20%) of the 144 adolescents had 2-LTR circles detected in PBMCs. None of the 14 children achieving virologic control by age one year had detectable 2-LTR circles compared with 15% and 27% of those achieving control between ages 1 and 5 years or after age 5 years, respectively (p=0.03). No association between 2-LTR circles and intermittent viremia was observed: 27 of the 29 (93%) adolescents with 2-LTR circles did not have intermittent viremia compared to 107 of the 115 (93%) adolescents with undetectable levels of 2-LTR circles (p=1.00). Furthermore, none of the 29 participants with 2-LTR circles were HIV negative or indeterminate compared with 21% of the 115 with undetectable levels of 2-LTR circles (p=0.004). The median proviral burden was significantly higher in the 29 adolescents with detectable 2-LTR circles (190.8 copies/million PBMCs, [IQR: 78.2, 257.6]) compared with 23.2 copies/million PBMCs (IQR: 5.1, 75.9) in the 115 adolescents without detectable 2-LTR circles (p<0.001).
Immune activation and long-term virologic control
Among the 9 pro-inflammatory cytokines analyzed, plasma concentrations of TNF-α, GM-CSF, IL-1β, IL-2 and IL-8 were substantially higher in PHIV+ than PHEU youth (Table 2). Only plasma IL-2 levels differed across the three PHIV+ groups, with levels higher in youth who suppressed HIV at younger ages. Concentrations of sCD14, a predictor of HIV disease progression and mortality in adults,17;18 did not differ by age at virologic control, but were significantly higher in PHIV+ than in PHEU adolescents (median 1.8 μg/mL vs. 1.3 μg/mL) (p<0.001, Table 2). Soluble CD163 concentrations were not significantly higher in PHIV+ than PHEU youth, (p=0.17, Table 2). Plasma markers of immune activation did not correlate with proviral DNA level or 2-LTR circle detection.
Table 2. Distribution of immune activation and inflammation markers by age at virologic control and among perinatally HIV-exposed but uninfected (PHEU) controls.
| Age at virologic control | |||||||
|---|---|---|---|---|---|---|---|
| Characteristic | Total PHIV+ (N=144) |
<1 year (N=14) |
1-5 years (N=53) |
>5 years (N=77) |
P-value† (PHIV+ cohorts) |
PHEU (N=10) |
P-value† (PHIV+ vs. PHEU) |
| Soluble CD14 (μg/ml) |
0.41 | <0.001 | |||||
| Median (Q1, Q3) |
1.8 (1.5, 2.1) |
1.9 (1.6, 2.3) |
1.8 (1.5, 2.0) |
1.8 (1.5, 2.1) |
1.3 (1.1, 1.5) |
||
| CD163 (ng/mL) | 0.20 | 0.17 | |||||
| Median (Q1, Q3) |
484.6 (375.3,641.6) |
448.6 (290.6, 554.2) |
481.1 (392.6, 695.4) |
510.0 (385.9, 632.8) |
376.3 (306.8, 549.6) |
||
| TNF-α (pg/mL) | 0.56 | <0.001 | |||||
| Median (Q1, Q3) |
21.9 (15.7, 28.7) |
19.6 (17.2, 25.7) |
21.9 (15.2, 26.7) |
22.9 (16.3, 29.3) |
11.3 (10.3, 14.1) |
||
| IFN-γ (pg/mL) | 0.88 | 0.95 | |||||
| Median (Q1, Q3) |
4.0 (3.5, 7.9) |
4.0 (3.0, 7.9) |
4.1 (3.7, 7.9) |
4.2 (3.7, 7.9) |
3.7 (3.7, 8.9) |
||
| GM-CSF (pg/mL) | 0.93 | <0.001 | |||||
| Median (Q1, Q3) |
2.5 (2.4, 2.9) |
2.6 (2.5, 2.6) |
2.6 (2.4, 2.9) |
2.5 (2.4, 2.9) |
1.0 (1.0, 1.0) |
||
| IL-1β (pg/mL) | 0.17 | 0.004 | |||||
| Median (Q1, Q3) |
3.7 (1.1, 7.9) |
2.0 (0.9, 3.7) |
3.3 (1.1, 6.9) |
3.7 (1.8, 9.2) |
1.1 (1.1, 1.1) |
||
| IL-2 (pg/mL) | 0.04 | <0.001 | |||||
| Median (Q1, Q3) |
1.9 (1.5, 2.4) |
2.4 (2.0, 2.4) |
1.9 (1.5, 2.4) |
1.9 (1.5, 2.4) |
0.9 (0.9, 0.9) |
||
| IL-6 (pg/mL) | 0.38 | 0.56 | |||||
| Median (Q1, Q3) |
2.5 (1.3, 4.8) |
2.5 (2.1, 3.0) |
2.3 (1.2, 4.0) |
3.0 (1.5, 5.2) |
2.5 (1.2, 3.5) |
||
| IL-8 (pg/mL) | 0.88 | 0.01 | |||||
| Median (Q1, Q3) |
18.5 (11.6, 26.5) |
18.4 (13.2, 29.2) |
18.6 (9.6, 27.9) |
18.2 (12.2, 23.4) |
8.9 (5.2, 14.7) |
||
| IL-10 (pg/mL) | 0.84 | 0.40 | |||||
| Median (Q1, Q3) |
6.4 (4.0, 11.1) |
6.8 (5.6, 7.9) |
5.4 (4.0, 10.9) |
7.0 (3.5, 13.5) |
6.9 (4.1, 28.0) |
||
| IL-12 (pg/mL) | 0.24 | 0.39 | |||||
| Median (Q1, Q3) |
3.3 (2.7, 5.8) |
2.9 (2.0, 5.9) |
3.3 (2.7, 5.8) |
3.9 (2.7, 5.8) |
2.7 (2.7, 2.7) |
||
= Kruskal-Wallis test
HIV Serologic Profiles in Perinatal HIV Infection under long-term cART
Five of the 14 children (36%) achieving control by age one year tested HIV seronegative by ELISA at their most recent visit, compared with 2 of 53 (4%) who achieved virologic control between 1 and 5 years of age and none of the 77 with virologic control after age 5 years. Younger age at virologic control was also associated with an indeterminate or negative HIV serostatus by Western blot, with 12 of 14 adolescents (86%) with virologic control before age one year testing indeterminate (N=10) or negative (N=2) compared with 10 of the 53 (19%) achieving virologic control between ages 1-5, and 2 of the 77 (3%) achieving virologic control after age 5 years (p<0.001). HIV serostatus was associated with proviral reservoir size. The median proviral load for 24 adolescents with indeterminate or negative HIV serostatus by Western blot was 4.3 copies/million PBMCs (IQR: 3.0, 18.3), which was significantly smaller than 56.7 copies/million PBMCs (IQR: 10.8, 150.1) in 120 adolescents with a positive Western blot profile (p<0.001) (Figure 3).
Figure 3.
Distribution of proviral load by HIV serostatus at last visit. The limit of detection for proviral load varies based on the number of cells available for analysis. Values of proviral load below the limit of detection were set to the limit of detection. Filled and open shapes represent samples tested above and below PBMC assay detection limit, respectively. Circles, squares, and triangles represent samples tested as western blot positive, negative, and indeterminate, respectively.
DISCUSSION
While current cART regimens are effective in controlling HIV replication to clinically undetectable levels for years, existing strategies do not eradicate replication-competent HIV reservoirs, including in perinatally-infected children. However, very early cART in perinatal infection may limit virus spread into HIV reservoirs and promote HIV remission or cure whereby viremic rebound does not recur when cART is stopped, as was demonstrated in the case of the “ Mississsippi Child”.13 Approaches to virus eradication in chronically HIV-infected persons in whom reservoirs are established are likely to be most effective in those who achieve marked restriction in proviral reservoir size on cART.19 Defining the virologic and immunologic endpoints following standard effective cART for perinatal infection and identifying factors and biomarkers associated with limited proviral reservoir size are critical to identify study participants for proof-of-concept studies aimed at curing HIV infection.
To our knowledge, this is the first cohort-based study to show that younger age at virologic control with standard cART is associated with marked reduction in proviral reservoirs in perinatally HIV infected children surviving to adolescence. Younger age at virologic control gave rise to a unique biomarker profile of small or undetectable peripheral blood HIV proviral load, absent 2-LTR circles, and indeterminate or negative HIV serostatus, suggesting excellent long-term control of HIV replication. The proviral reservoir size was also smaller when virologic control occurred between 1- 5 years of age compared with after age 5, emphasizing the impact of earlier treatment on proviral reservoir size in perinatal infection. This difference in viral reservoir size as a function of age at virologic control fits with the developmental aspects of memory CD4+ T cell formation during childhood, whereby memory CD4+ T cells develop slowly over the time course of years.20;21 Infants are born with few circulating memory CD4+ T cell compartment (<10%) compared with adults (50%). Furthermore, long-lived central memory CD4+ T cells, the predominant reservoir for HIV,22 are limited in infants, exemplifying the interaction between early virologic control and eventual reservoir size. This is consistent with time to first undetectable viral load being strongly correlated with the size of the resting CD4+ T-cell latent HIV reservoir at two years of age in perinatal infection.23 It is, however, important to emphasize that undetectable levels of HIV provirus in peripheral blood likely still allow for the presence of replication-competent HIV that can rekindle viremic rebound if cART is stopped. The limitation in the current use of undetectable proviral DNA to assess HIV clearance and cure is highlighted by the two Boston patients who achieved undetectable proviral DNA levels following bone marrow transplantation, yet rebounded after cART was discontinued 24 The encouraging single case of the Mississippi Child13 prompts the need for a more robust analysis of a larger cohort treated shortly after birth.
Proviral DNA levels are thought to be fairly stable following the first year of cART,9;25;26 despite ongoing treatment. Replication-competent HIV reservoirs also show minimal decay over long-term cART.27 However, more recent studies in adults25 and children suggest that some decay in HIV proviral DNA levels continues over 10-15 years of long-term follow-up.28 In four PHIV+ youth who initiated cART at a median age of 2 months, decay of proviral load continued over a median of 14 years of virologic suppression, reaching an extraordinarily low level (median: 0.86 log10 copies/million PBMC).28 This level is substantially lower than the median proviral load of 2.15 log10 copies/million PBMCs reported in adults initiating cART during acute HIV infection followed by a median of 10 years of virologic suppression,25 although different techniques were used to measure HIV proviral burden. In the present cross-sectional study, we were unable to assess the independent effects on proviral load of duration of virologic control and age at virologic control, which were highly correlated. Ongoing proviral decay under long-term cART in PHIV+ individuals thus warrants further study. Defining the interaction between age at virologic control, duration of suppression, and decay of peripheral blood HIV reservoirs under long-term cART in perinatal infection is a critical next step for refining therapeutic strategies aimed at achieving HIV remission or cure for this population.
Immune activation as a consequence of HIV infection is a known contributor to HIV-related morbidity and mortality.29 Treatment during acute HIV infection in adults decreases, but does not reverse, abnormal immune activation.8 Levels of sCD14, a biomarker of monocyte activation, result from microbial translocation through the intestinal mucosa during HIV infection.30 Microbial translocation is also increased in early infancy in perinatal HIV infection.31 In our study, levels of sCD14 remained abnormally high despite long-term virologic control, even in the children who achieved virologic control by age one year with small proviral reservoirs. Studies of the immunopathogenesis of chronic immune activation in patients with extremely small proviral burden, as reported here, should provide additional insights into this process and suggest that the initial gut-associated abnormalities induced by HIV infection32;33 are not restored with long-term virologic control.
The contribution of ongoing virus replication to the maintenance of proviral reservoirs under cART is controversial. Despite prolonged virologic control (median follow-up of 7.2 years), 2-LTR circles were detected in 20% of the study participants and were associated with more than 8-fold larger proviral reservoirs (190 vs 23 copies/copies PBMCs), suggesting ongoing replenishment of HIV reservoirs in a subset of patients despite seemingly effective virologic control on cART. Importantly, none of the study participants who achieved virologic control by age one year had detectable levels of 2-LTR circles.
Early effective control of HIV replication has been associated with incomplete development of HIV-specific immune responses.4;10;32;34;35 The phenomenon of absent HIV-specific antibody responses in PHIV+ children treated with early, effective cART has been reported in up to 94% of such children when tested at a median age of 21 months4 and between 47-66% of those tested between ages 5 and 6 years.10;36 Seventy-five percent of PHIV+ children with effective virologic control from infancy for a median of 5.5 years tested negative or indeterminate for HIV but still harbored replication-competent HIV in the resting memory CD4+ T cell reservoir.36 The lack of HIV-specific antibodies in this setting likely reflects early control of virus replication with subsequent limited exposure to HIV antigens. We found significantly smaller proviral load among youth who tested seronegative or indeterminate for HIV. Seronegative or indeterminate HIV status may therefore provide a useful biomarker for small proviral reservoirs in perinatal infection.
Limitations of this study include lack of analyses of replication-competent proviral reservoirs that require large blood volumes and freshly collected blood in addition to longitudinal data to fully assess the contribution of ongoing proviral reservoir decay to low proviral burden under long-term cART for perinatal HIV infection. Analyses of genetic factors that may contribute to long-term virologic control or small reservoir size, such as HLA-alleles or heterozygosity of the CCR5δ32 mutation, were not performed. Nevertheless, this is the first study to comprehensively analyze the virologic and immunologic effects of long-term cART on biomarkers of HIV persistence in a large number of PHIV+ youth, with the identification of unique patterns of persistence based on age at virologic control.
The World Health Organization recommends cART in all HIV-infected children under five years old. Initiating antiretroviral treatment within the first few days of life in HIV infected neonates, as in the child from Mississippi,13 to achieve virologic remission or cure in perinatal infection is currently challenging due to obstacles in early infant diagnosis and antiretroviral drug options for this age group.37 With cART, PHIV+ children have the potential to survive to adulthood.38 This study adds evidence that early effective control of virus replication in perinatal infection with cART, along with prolonged virologic suppression, leads to substantially restricted HIV peripheral blood proviral reservoirs and makes these children and adolescents potential candidates for interventions examining HIV remission or cure.
ACKNOWLEGMENTS
Drs Persaud and Patel had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
We thank the children and families for their participation in PHACS, and the individuals and institutions involved in the conduct of PHACS. We thank Linda Lambrecht and Margaret McManus from the University of Massachusetts for their contribution to the HIV serologic testing and Matt Strain and Steven Lada from the University of California San Diego for their advice and support on proviral DNA quantitation.
The laboratory studies were supported by grants from the NIH (AI93701 and HD0577849 to Dr. Persaud) and by funding from the American Foundation for AIDS research (to Drs. Persaud and Luzuriaga); by grants from the International Maternal Pediatric Adolescent AIDS Clinical Trials Network (UO1-AI-068632 to Dr. Persaud). Dr. Richman was supported by the Collaboratory for AIDS Research on Eradication (CARE; U19 AI096113), the UCSD Center for AIDS Research (AI306214), and the James Pendleton Charitable Trust.
The study was also supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development with co-funding from the National Institute on Drug Abuse, the National Institute of Allergy and Infectious Diseases, the Office of AIDS Research, the National Institute of Mental Health, the National Institute of Neurological Disorders and Stroke, the National Institute on Deafness and Other Communication Disorders, the National Heart Lung and Blood Institute, the National Institute of Dental and Craniofacial Research, and the National Institute on Alcohol Abuse and Alcoholism, through cooperative agreements with the Harvard University School of Public Health (HD052102, 3 U01 HD052102-05S1, 3 U01 HD052102-06S3) (Principal Investigator: George Seage; Project Director: Julie Alperen) and the Tulane University School of Medicine (HD052104, 3U01 HD052104-06S1) (Principal Investigator: Russell Van Dyke; Co-Principal Investigator: Kenneth Rich; Project Director: Patrick Davis). Data management services were provided by Frontier Science and Technology Research Foundation (PI: Suzanne Siminski), and regulatory services and logistical support were provided by Westat, Inc (PI: Julie Davidson).
The following institutions, clinical site investigators, and staff participated in conducting PHACS/AMP in 2012, in alphabetical order: Baylor College of Medicine: William Shearer, Mary Paul, Norma Cooper, Lynette Harris; Bronx Lebanon Hospital Center: Murli Purswani, Mahboobullah Baig, Anna Cintron; Children’s Diagnostic & Treatment Center: Ana Puga, Sandra Navarro, Doyle Patton, Deyana Leon; Children’s Hospital, Boston: Sandra Burchett, Nancy Karthas, Betsy Kammerer; Ann & Robert H. Lurie Children’s Hospital of Chicago: Ram Yogev, Margaret Ann Sanders, Kathleen Malee, Scott Hunter; Jacobi Medical Center: Andrew Wiznia, Marlene Burey, Molly Nozyce; St. Christopher’s Hospital for Children: Janet Chen, Latreca Ivey, Maria Garcia Bulkley, Mitzie Grant; St. Jude Children’s Research Hospital: Katherine Knapp, Kim Allison, Megan Wilkins; San Juan Hospital/Department of Pediatrics: Midnela Acevedo-Flores, Heida Rios, Vivian Olivera; Tulane University Health Sciences Center: Margarita Silio, Medea Jones, Patricia Sirois; University of California, San Diego: Stephen Spector, Kim Norris, Sharon Nichols; University of Colorado Denver Health Sciences Center: Elizabeth McFarland, Emily Barr, Robin McEvoy; University of Medicine and Dentistry of New Jersey: Arry Dieudonne, Linda Bettica, Susan Adubato; University of Miami: Gwendolyn Scott, Patricia Bryan, Elizabeth Willen.
Funding/Support: The Pediatric HIV/AIDS Cohort Study (PHACS) was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development with co-funding from the National Institute on Drug Abuse, the National Institute of Allergy and Infectious Diseases, the Office of AIDS Research, the National Institute of Mental Health, the National Institute of Neurological Disorders and Stroke, the National Institute on Deafness and Other Communication Disorders, the National Heart Lung and Blood Institute, the National Institute of Dental and Craniofacial Research, and the National Institute on Alcohol Abuse and Alcoholism, through cooperative agreements with the Harvard University School of Public Health (HD052102, 3 U01 HD052102-05S1, 3 U01 HD052102-06S3) and the Tulane University School of Medicine (HD052104, 3U01HD052104-06S1).Note: The conclusions and opinions expressed in this article are those of the authors and do not necessarily reflect those of the National Institutes of Health or United States Department of Health and Human Services.
Footnotes
Disclosures: Dr. Richman has consulted for Chimerix, BMS, Gilead, Gen-Probe, Monogram, Sirenas, Prism.
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