Age at ART initiation and proviral reservoir size in perinatal HIV-1 infection: considerations for ART-free remission

Purpose of review Achieving ART-free remission without the need for lifelong antiretroviral treatment (ART) is a new objective in HIV-1 therapeutics. This review comprehensively examines the literature to evaluate whether the age at ART initiation in children with perinatal HIV-1 influences the size and decay of the HIV-1 reservoir. The insights gathered from this review serve to inform the field on the unique dynamics of HIV-1 reservoir size in perinatal HIV-1 infection as a function of age at ART initiation, as well as inform biomarker profiling and timing of ART-free remission strategies for children living with HIV-1 globally. Recent findings Recent studies demonstrate that initiating very early effective ART in neonates is feasible and limits HIV-1 reservoir size. The clinical relevance of limiting the HIV-1 reservoir size in perinatal infection was recently demonstrated in the Tatelo Study, which investigated a treatment switch from ART to two broadly neutralizing antibodies (bNAbs) in very early treated children. Low proviral reservoir size was associated with sustained virologic control for 24 weeks on bNAbs. Summary Immediate and early ART initiation for neonates and infants with perinatal HIV-1 is essential to restricting HIV-1 reservoir size that may enable ART-free remission.


INTRODUCTION
Children and adolescents living with perinatal HIV-1 remain an under-researched population in the field of HIV-1 reservoir dynamics [1].Of the 1.7 million children living with perinatal HIV-1 worldwide, only 52% are receiving lifesaving, effective combination antiretroviral treatment [2].Without ART, approximately one-third of children living with perinatal HIV-1 die within the first year of life [3,4] while 50% do not survive past the age of 2 [5].International guidelines now recommend that all individuals with a confirmed HIV-1 diagnosis should initiate ART immediately, irrespective of age, immunological, or clinical status [6].In the context of perinatal HIV-1, early initiation of ART within the 3 months following birth has been established as lifesaving standard of care in many settings [7,8].As in adults, the majority of children living with HIV-1 experience viral rebound within weeks of ART interruption, including those who previously achieved long-term viral suppression [1,9,10].
The effects of age at ART initiation on proviral reservoir size remain poorly understood.The most studied biomarker to approximate the reservoir size and dynamics in the context of perinatal HIV-1 infection is total concentrations of cell-associated HIV-1 DNA; multiple studies support that early ART leads to lower proviral loads later in childhood that are maintained through adolescence and adulthood [11 regards to perinatal HIV-1 and potential for ART-free remission.Discerning differences of age at ART initiation is crucial for optimizing the design and delivery of effective remission and cure strategies.This report seeks to answer these questions through a systematic review of the literature on ART initiation and proviral reservoir size in perinatal HIV-1, including recent reports from very early treated neonates.

KEY POINTS
Accelerated diagnosis and immediate antiretroviral therapy (ART) initiation for infants with perinatal HIV-1 is essential to restricting the reservoir size in order to enable ART-free remission The clinical relevance of achieving low reservoir size in perinatal HIV-1 infection through early and very early ART is supported by the three pediatric cases of ARTfree remission reported to date.
Findings from the Tatelo Study support that low reservoir size achieved through very early ART of neonates may afford treatment switch to combination therapy with monthly infusions of two broadly neutralizing antibodies, which paves the way towards long-acting biologics for improved sustained virologic suppression in children.per million (cpm) PBMCs at age of treatment initiation with a median cohort size of 12  published between 2000-2023 were reviewed.Publications reporting total HIV-1 DNA were stratified by treatment timing: late treatment (ART initiated >3 months of age; n ¼ 19); early treatment (ART initiated >7 days to 3 months of age; n ¼ 13); and very early treatment (ART initiated 7 days of life; n ¼ 9).Search criteria and methods are further explained in the Supplemental Methods, Supplemental Digital Content, http://links.lww.com/COHA/A18.
In the 19 publications reporting on late treatment, ART was initiated at a median of 9.6 months, spanning 4-154.9months of age with baseline HIV-1 DNA ranging from 2.9 to 4 log 10 cpm PBMCs.The lowest total HIV-1 DNA reported following late ART was 1.3 log 10 cpm PBMC at approximately 12.9 years of age [15,16] with a median age of ART initiation of 1.9 years [15] and 9 years [16].Zanchetta et al. [16] followed 14 children who initiated ART at a median age of 9 years with a baseline HIV-1 DNA of 2.9 log 10 cpm PBMC.In contrast, at median 9.8 years of age, Brice et al. [17] found the median HIV-1 DNA was 2.6 log 10 cpm PBMC in a cross-sectional study of 97 virologically suppressed children who initiated ART at a median 3.3 years of age.Overall, HIV-1 DNA varied greatly in late treated children and declined the least with increasing age and duration of ART (Fig. 2a, Fig. 3).
Of the 13 publications reporting on early ART initiation, ART was initiated at a median of 2 months, spanning 1.7-3 months of age with baseline DNA ranging from 3 to 3.4 log 10 cpm PBMC (Fig. 2b, Fig. 3).The lowest median total HIV-1 DNA reported following early ART initiation was 0.6 log 10 cpm PBMC at 12.6 years with a median ART initiation age of 2.4 months in one study [15].The Children with Early Antiretroviral Therapy (CHER) trial found early treatment with sustained long-term ART resulted in reduced levels of HIV-1 DNA when compared to children treated later and in those who underwent analytical treatment interruption (ATI) in the parent trial [7,11  & ,17].Specifically, HIV-1 ].After ART reinitiation following ATI, there was no significant difference HIV-1 DNA between treatment initiation groups at 248 weeks on ART, indicating benefits of early treatment may be lost without continuous ART.In a follow up, Katusiime et al. [18] investigated the proviral landscape of a small cohort of early and late-treated children from the CHER trial after 6-9 years of ART.Interestingly, genetically intact HIV-1 sequences were only identified in late treated children, suggesting early ART may lead to preferential deletion of intact proviruses.
Multiple studies directly compared HIV-1 DNA after early or late ART initiation [11 & ,15,17 -24].In a small cohort, Luzuriaga et al. [19] compared HIV-1 DNA decay in infants who initiated ART between 0.5-2.6 months of age and to those treated between 12.3 and 14.5 years after established infection.HIV-1 DNA decayed below 1.1 log 10 cpm PBMC by 14-17.7 years of age in the early treated group whereas HIV-1 DNA ranged from 1.8 to 2.5 log 10 cpm PBMC by 19-23 years of age in the late treated group.This trend is further demonstrated across all studies analyzed (Fig. 3); while HIV-1 DNA continues to decline in both groups, early treated children reach and maintain lower HIV-1 DNA levels over time.
Uprety et al. [21] investigated the long-term decay dynamics of HIV-1 infected cells in children living with perinatal HIV-1.Virologic suppression achieved by 1 year of age following early ART was associated with faster and greater decline in total HIV-1 DNA 2 years following virologic suppression though no significant difference in decay dynamics was observed between 2-14 years of ART.Despite this, early treated children achieved significantly lower HIV-1 DNA levels after 10 years of ART, reaching 1.0 log 10 cpm PBMC in contrast to late treated children with 1.5 log 10 cpm PBMC [21].Similarly, McManus et al. [20] observed total HIV-1 DNA levels in early treated children trended lower than late treated children, though levels did not reach statistical significance.
In the nine publications reporting on very early ART, treatment was initiated at a median of 32 h, spanning 7 h-6 days of life with baseline HIV-1 DNA ranging from 2.4 to 3-log 10  The EIT study enrolled 40 infants in Botswana who initiated ART at <7 days of age; only 38 children survived until week 24 [25].García-Broncano et al. [25] reported median total HIV-1 DNA to be 0.7 log 10 cpm PBMC at a median age of 20.7 months for 10 of the 40 children who initiated ART at a median 7 h after birth.This value is contrasted to a group of 10 control children with a median total HIV-1 DNA of 3.0 log 10 cpm PBMC at a median age of 25.4 months who initiated ART at a median age of 4 months and a group of adults living with chronic HIV-1 with a median total HIV-1 DNA of 2.1 log 10 cpm PBMC after a median of 16 years on ART.In a follow up with all 38 infants, Ajibola et al. [28] reported a median HIV-1 DNA of 1.9 log 10 cpm PBMC by 84-96 weeks after ART initiation at a median of 2 days after birth.By week 96, 74% (28/38)  ] followed 164 South African neonates with in-utero HIV-1 who initiated ART at a median age of 6.5 days.98/164 (60%) neonates achieved virologic suppression within 6 months of life.In the subgroup of 98 infants who achieved virologic suppression, 40/274 HIV-1 DNA measurements fell below the limit of detection (LOD) of 25 cpm PBMC in the first 3.5 years of life.In this cohort, male sex was correlated with smaller reservoir size, with male infants achieving HIV-1 DNA levels 0.5 1og 10 cpm PBMC lower than female infants.In addition, lower baseline plasma viral load and higher baseline CD4 þ T-cell percentage in the neonates garnered the strongest association with smaller reservoir size.
The LEOPARD study conducted in South Africa followed 63 neonates who initiated ART at median 2 days postbirth with baseline HIV-1 DNA of 3.2 1og 10 cpm PBMC.After 48 weeks on ART, infants who initiated ART at <48 h and 49 h-14 days declined to a median 2.4 1og 10 cpm PBMC while those who initiated ART >14 days of life maintained HIV-1 DNA levels of 3.2 1og 10 cpm PBMC.This study found that smaller reservoir size in infants was associated with higher CD4 þ T-cell percentage and viral load <100 000 copies/ml pre-ART [ [31].In line with sex differences observed in Millar et al., in Cohort 1, female sex was associated with greater risk of virologic failure.Altogether, these studies have established that accelerated diagnosis and immediate ART initiation in neonates with perinatal HIV-1 is essential to restricting the reservoir size with the potential for enabling ART-free remission.These observations are exemplified when comparing the HIV-1 DNA trends over time in all studies analyzed (Figs.2c and 3).
The studies analyzed were highly variable with respect to median ART initiation age, sample size, follow-up time of subgroups, and different HIV-1 DNA assays.Nevertheless, earlier ART initiation was associated with lower HIV-1 DNA over time in all studies (Figs. 2 and 3).Further, very early ART initiation has the potential to achieve exceedingly low levels of HIV-1 DNA, reaching concentrations <0.6 log 10 HIV-1 DNA cpm PBMC by 2 years of age.Thus, very early ART presents the opportunity for ART-free remission if immune-based therapies that control HIV-1 rebound off ART are identified.

IMPACT OF HIV-1 DNA ON ART-FREE REMISSION AND POTENTIAL CURE STRATEGIES IN PERINATAL HIV-1
Case studies on ART-free remission in perinatal HIV-1 infection with very early ART initiation such as the 'Mississippi baby' and early, time-limited ART in the 'French adolescent' and 'South African boy' have demonstrated the potential for maintaining sustained virologic in the absence of ART in children [32][33][34][35].However, unexpected viral rebound, as evidenced by the case of the Mississippi baby [36], indicates that HIV-1 reservoirs are established early in the course of HIV-1 infection with sustained quiescence for over 2 years of ART.Combinations of interventions that restrict HIV-1 reservoirs as with very early ART initiation with immune-based therapies will likely be necessary to achieve lifelong ARTfree remission.
One area of research currently under investigation has been the use of broadly neutralizing antibodies (bNAbs) in combination with ART.The Tatelo Study recently investigated the safety and efficacy of combination bNAbs, VRC01LS and 10-1074, in very early treated children with HIV-1.This study provides proof-of-concept of the potential for these bNAbs to be used for sustained virologic control [37,38 && ].The Tatelo study focused on a subset of 25 children originally enrolled in the EIT study who initiated very early ART within 7 days postbirth.These children were administered dual bNAb monthly infusions for either 2 (n ¼ 6) or 8 months (n ¼ 19), followed by bNAb treatments continued in the absence of ART for up to 6 months (n ¼ 25).While 56% (14/25) experienced viral rebound within 4 weeks after ART discontinuation, 44% (11/25) maintained viremia below 400 copies of HIV-1 RNA/mL plasma for the 6-month timeframe.When evaluating biomarkers that correlated with successful bNAb response, the researchers identified that total HIV-1 DNA levels near birth was significantly lower in the children that responded successfully to bNAb treatment.Thus, the findings of the Tatelo study indicate that bNAbs may represent a potential treatment strategy for sustained virologic suppression in the setting of low proviral loads in the context of perinatal HIV-1.
Therapeutic approaches to reactivate and eliminate latent HIV-1 reservoirs to achieve HIV-1 remission are also under investigation in adults living with HIV [1].Recent studies [39][40][41][42][43] have emphasized the need for a comprehensive understanding of potential differences in reactivation based on characteristics including age, sex, and resource setting.Identification of virological and immunological biomarkers predictive of ART-free remission are also crucial to advancing the field of HIV-1 cure research for a diverse population [44].Perinatal HIV-1 offers a distinct opportunity to investigate the efficacy of HIV-1 remission interventions in relation to the timing of both transmission event, ART initiation and remission therapeutics.

LIMITATIONS OF TOTAL HIV-1 DNA MEASUREMENTS
In the studies reviewed, the viral reservoir was largely quantified as total HIV-1 DNA cpm PBMC, a measurement that overestimates the size of the replication-competent HIV-1 reservoir due to an abundance of defective proviruses [45].Use of total HIV-1 DNA in PBMCs can provide a near maximal estimate of the concentration of HIV-1-infected cells in the circulation for which an overall estimate of infectious reservoir size can be derived.
The Quantitative Viral Outgrowth Assay (QVOA) is considered the gold standard for quantifying the induced replication-competent reservoir [44].However, this assay often requires higher blood volumes that cannot feasibly be extracted from pediatric populations living with HIV-1.Further, this assay may also require viable cells unattainable in some resource-constrained settings.The Intact Proviral DNA Assay (IPDA) allows for the differentiation between intact and defective genomes, increasing accuracy with minimal required blood volume.Studies in adults living with HIV-1 have demonstrated the heightened decay of cells harboring replication-competent intact proviruses and potentially replication-competent over those harboring defective proviruses [46,47].In both adult and pediatric populations, intact proviruses levels positively correlate with total HIV-1 cpm CD4 þ T cells [46,48].The advent of newer multiplex HIV-1 DNA assays optimized for different HIV-1 subtypes will be instrumental for identifying the composition of the proviral pool capable of eliciting rebound after ATI.

CONCLUSION
This systematic review focused on understanding the impact of age at ART initiation on total concentrations of HIV-1-infected cells in PBMCs.Despite similar pre-ART baseline HIV-1 DNA levels, very early treatment results in lower levels of HIV-1 DNA in early childhood.More research is necessary to understand optimizing clearance and control of HIV-1 reservoirs in perinatal HIV-1 infection.The potential correlation of additional factors, such as sex differences, geographic region of residence, HIV-1 subtypes, with HIV-1 viral reservoir formation and stability should be further investigated.

REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest

FIGURE 1 .
FIGURE 1. Venn diagram illustrating the intersection of the 26 papers examining total HIV-1 DNA in perinatal HIV-1, categorized by the timing of ART initiation --very early, early, and late.The diagram highlights the overlap and distinctions in studies focusing on these specific treatment initiation groups.ART, antiretroviral treatment; HIV-1, human immunodeficiency virus-1.

FIGURE 2 .
FIGURE 2. Median log 10 HIV-1 DNA cpm PBMCs measurements since initiation of ART from the 26 studies reporting on (a) late, (b) early, and (c) very early ART initiation.ART, antiretroviral treatment; cpm, copies per million; HIV-1, human immunodeficiency virus-1; PBMC, peripheral blood mononuclear cell.

FIGURE 3 .
FIGURE 3. Bubble plot illustrating median log 10 HIV-1 DNA cpm PBMCs measurements across age in months from a total of 26 studies reporting on very early (orange), early (pink), or late (blue) ART initiation.This visual representation provides insights into the variations in HIV-1 DNA levels among different treatment initiation categories and age at measurement, with the bubble size reflecting sample size within the reported data.ART, antiretroviral treatment; cpm, copies per million; HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cell.