A child with perinatal HIV infection and long-term sustained virological control following antiretroviral treatment cessation

Understanding HIV remission in rare individuals who initiated antiretroviral therapy (ART) soon after infection and then discontinued, may inform HIV cure interventions. Here we describe features of virus and host of a perinatally HIV-1 infected child with long-term sustained virological control. The child received early limited ART in the Children with HIV Early antiRetroviral therapy (CHER) trial. At age 9.5 years, diagnostic tests for HIV are negative and the child has characteristics similar to uninfected children that include a high CD4:CD8 ratio, low T cell activation and low CCR5 expression. Virus persistence (HIV-1 DNA and plasma RNA) is confirmed with sensitive methods, but replication-competent virus is not detected. The child has weak HIV-specific antibody and T cell responses. Furthermore, we determine his HLA and KIR genotypes. This case aids in understanding post-treatment control and may help design of future intervention strategies.

R apid formation of persistent viral reservoirs follows acute HIV-1 infection. This early establishment of latently HIV-1-infected CD4+ T cells harbouring replication-competent virus remains the major obstacle to HIV cure or remission [1][2][3] . As antiretroviral therapy (ART), even when given within days of infection, usually fails to clear these reservoirs [4][5][6] , it is unlikely that ART alone can lead to HIV remission. It is, however, hypothesized that ART given very soon after infection may enable a more effective immune response and, together with other strategies, lead to sustained control of viral replication.
Current approaches to HIV cure or remission have focused on either reversing latency (e.g. "shock and kill"), enhancing immune responses or preventing immune activation (e.g. vaccines and other immunotherapies) 7 . Central to the question of HIV remission is the interaction between viral reservoir, immune activation, host genetics and immune response.
In children, data are extremely limited. In 2013, the report of the "Mississippi baby" suggested that very early ART, here within 30 h of birth, could lead to prolonged (27 months) virological control off-treatment 19,20 , raising hope for a feasible HIV-1 remission strategy. Unfortunately, this girl "relapsed" after almost 2 years without ART due to return of high levels of viral replication, and required ART. Subsequently, a French girl was reported who started ART at 3 months of age, stopped treatment between 5 and 7 years of age and controlled virus to undetectable levels for over 12 years 21 .
Reports of post-treatment controllers who initiated ART and then discontinued by design or unintentionally may help our understanding of key host determinants of HIV replication control, and inform interventions for HIV remission and cure.
Here we report a detailed virological and immunological analysis of a child at 9.5 years of age, originally enroled in the Children with HIV Early antiRetroviral therapy (CHER) trial 22,23 who was randomized to the immediate, time-limited 40 weeks of ART study arm. The CHER trial was initiated at a time when the best strategy on when to initiate and how to maintain treatment in infants was unclear. This child, one of 227 early treated children (0.4%), is the only one maintaining long-term sustained virological control post-ART cessation. At 9.5 years, virus persists at low levels (plasma RNA 6.6 copies per mL), cell-associated DNA is 5 copies per million peripheral blood mononuclear cells and replication-competent virus is not detected. Immunologically, he is not unlike healthy children of similar age, evidenced by high CD4:CD8 ratio, low T cell activation and low CCR5 expression. He has weak HIV-specific antibody and CD4+ T cell responses indicating memory of prior/current virus encounter, and together with possession of some host genotypes, these provide clues for future studies to inform what constitutes long-term post-treatment control.

Results
Clinical case. The child, born in 2007, had a positive HIV-1 DNA PCR at age 32 days. At 39 days, HIV-1 RNA was >750,000 copies per mL (upper limit of quantitation of the assay) confirming infection; at 60 days, plasma HIV RNA had declined to 151,000 copies per mL. He commenced zidovudine, lamivudine and lopinavir-ritonavir one day later (Fig. 1, Supplementary Table 1). He was born at term, of normal birth weight (3700g), did not receive nevirapine prophylaxis, and was not breastfed. CD4+ T cell count and per cent at 61 days, prior to ART start, were 2249 cells per µL and 41.6%. These values fell within the respective baseline interquartile ranges (IQRs) for all early treated children who stopped ART in the CHER trial (Supplementary Table 2)n = 227 children; median CD4+ T cell count was 2255 (IQR: 1759-2972); median CD4% 36.4 (IQR: 31.4-42.5).
Viral load (VL) declined to <50 RNA copies per mL after 24 weeks of ART. At 50 weeks of age, when treatment was stopped per trial protocol, VL was <20 copies per mL. Thereafter, VL remained below detection over 8.75 years without ART (Fig. 1a). CD4+ T cell counts remained normal-for-age (Fig. 1b) and CD4% remained above 30% throughout (Fig. 1c). At 9.5 years plasma drug concentrations for the most commonly used antiretroviral agents in South Africa were undetectable. The mother's CD4+ T cell count was 108 cells per µL when he was 7 months of age, and then 129 cells per µL 20 months later-these are the only maternal data available.
Virus persistence. At 9.5 years, the Roche VL result was reported as target not detected (TND; Fig. 2a). Virus pelleted from 10 mL of plasma, yielded 66 copies and a VL of 6.6 RNA copies per mL (Fig. 2a). Using a highly sensitive RNA nested qPCR on 3 mL of plasma, 2 of 22 replicates were positive (Fig. 2a). Using a seminested real-time PCR (sn-qPCR) assay and an input of 1 µg of genomic DNA (gDNA) per well, total cell-associated HIV-1 DNA was estimated at 5 copies per 10 6 peripheral blood mononuclear cells (PBMCs; six of nine amplifications positive) (Fig. 2b). In a stored sample from ART interruption at 50 weeks of age, this was almost identical (5 copies per 10 6 PBMCs: 1 of 3 amplifications positive). DNA sequencing of gag from the 9.5 year sample confirmed infection with subtype C virus (Fig. 2c). Using two virus outgrowth assays (primary CD8-depleted PBMCs and MOLT4/CCR5 cells), no replication-competent virus was detected at 9.5 years (Fig. 2d). However, a weak HIV RNA signal (1 of 24 replicates was positive) was detected in the day 28 supernatant of cultured primary CD4+ cells from the 50-week sample using qualitative real-time PCR nested assay (n-qPCR). The child's CD4+ T cells could be infected in vitro with the HIV-1 BaL strain (Fig. 2d).
Immunophenotyping. The CD4:CD8 T cell ratio was 1.9, higher than all uninfected control children (Fig. 5a). Measurement of T cell subsets representing various stages of differentiation highlighted that 9.5-10-year-old HIV-uninfected children do not display adult-like proportions of the different subsets, particularly for naive, central memory and effector memory CD4+ T cell subsets and central memory CD8+ T cells (Fig. 5b). The child had high proportions of naive, central memory and effector memory CD8+ T cells compared to uninfected children.
CCR5 density on CD4+ and CD8+ T cells was similar for children and adults, with the child having lower CCR5 expression than 14 of the 15 uninfected children/adults (Fig. 5c). Proportions of the child's CCR5-expressing CD4+ and CD8+ T cells were comparable to those of uninfected children. Of note, CCR5expressing CD8+ T cells were significantly higher in adults than . The X-axis shows age in weeks and then in years, separated by a blue vertical dotted line. Note: the detection limit of the VL assays used was 400 RNA copies per mL at 10.71 and 116.71 weeks; 50 RNA copies per mL at 33.71 weeks and 20 RNA copies per mL at all other time points. ART antiretroviral therapy NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08311-0 ARTICLE children (p = 0.0013; Mann-Whitney U test). Levels of immune activation, measured by HLA-DR, were similar to those of uninfected children and adults. Expression of TIGIT in the child's CD4+ and CD8+ T cells was similar to uninfected children, and significantly higher in adults (p = 0.027 and p = 0.0027, respectively; Mann-Whitney U tests). PD-1 expression on the child's CD4+ T cells was substantially above the median values for children or adults. However, two adult outliers had similarly high levels. Similar to TIGIT, PD-1 expression on CD8+ T cells was significantly higher in adults than in children (p = 0.0007; Mann-Whitney U test), with the child having high levels similar to the adult median. Flow cytometry data are provided in Supplementary Table 3.

Discussion
We report virological and immunological characteristics in a South African child of 9.5 years of age in long-term HIV remission. He initiated ART at 8.7 weeks of age and discontinued ART after 40 weeks in accordance to his randomization in the CHER trial 22 . This is the only child to achieve this outcome among 227 who stopped ART (at 40 or 96 weeks) in the trial (0.4%). The French case of long-term remission was one of 15 children in the French paediatric cohort who stopped ART (6.7%) 21 .
Studies have shown that earlier ART initiation results in a smaller HIV reservoir size [26][27][28] . Treatment was very early (within 30 h of birth) for the Mississippi baby who achieved 27 months of virological control off-treatment before experiencing virological rebound 19,20 . This delay in rebound may have been attributed to a small size of latent replication-competent reservoir. Both the French case with >12 years of remission 21 and this South African case started ART later, at 3 and 2 months, respectively. HIV transmission was likely intrapartum in the French child and in utero in the Mississippi baby. The timing is unknown for the South African child. Timing of transmission may have been a key factor affecting the different outcomes of remission. Of note, subtype of virus (B, H and C, respectively), treatment duration (18 months, 6 years, 10 months, respectively) and ethnicity were different in these children. Both earlier cases were girls, while this is the first report of a boy with HIV remission. Unlike the South African child, using similar methods and number of CD4+ T cells, replication-competent virus was readily found in the French case 21 . In contrast, no replication-competent virus was found in the Mississippi baby when testing 22 million CD4+ T cells 19 , highlighting that this measure poorly predicts long-term likelihood of remission. Furthermore, recent reports of very early treatment in adult cases (within 2 weeks of infection) described multiple sensitive tests for HIV-1 persistence, which supported the absence of detectable virus during ART-these cases all rebounded shortly after ART was stopped (n = 8 patients 5 ; median 26 days, range 13-48 days; n = 1 patient 6 , 225 days). Collectively, these findings suggest some limited viral replication may in fact be required for durable long-term remission. Early in infection the child's VLs were high, indicative of a highly replicating virus. The VL declined from >750,000 to 150,000 RNA copies per mL prior to beginning treatment, suggesting an immune response attempting to control virus replication. After ART initiation, viral decline was biphasic, with the expected initial sharp decline followed by a more gradual decline. After ART cessation, VLs remained below the detection threshold for 8.75 years.
More sensitive methods for VL measurement confirmed the presence of low amounts of virus produced in vivo (6.6 RNA copies per mL). Inability to detect replication-competent virus in vitro may be because of assay sensitivity (2 million CD4+ cells tested), or defective virus that cannot accumulate to detectable levels.
Early initiation of ART is associated with non-reactive HIV antibody results in many HIV-1-infected children and adults [29][30][31] . On standard assays the child is seronegative; however, results from bead arrays revealed footprints of historical adaptive responses that have either waned or are being maintained through ongoing antigenic priming. The substantial IgA2 (mucosal) response to gp41 could be primed by microbial antigens sharing homology with gp41 [32][33][34] . However, a recent study reported stronger gp41-specific IgA responses in elite controllers, which could not be well explained by responses to microbial antigens 35 . HIV-1 Env-and Vpu-specific NK cell antibodydependent cellular cytotoxicity (ADCC) responses, including against C-terminal peptide Vpu19 (our Vpu9 peptide), are associated with elite HIV-1 control 36 . Using a whole blood assay, we have shown similar responses associated with reduced maternal-infant HIV-1 transmission and lower VLs in HIV-1infected mothers 37,38 . The child lacked a detectable NK cell response to any HIV-1 peptide pool, likely due to very low levels of antibodies that target Env and Vpu. The Vpu9-IgM antibody response suggests a potential for interaction with complement. Anti-Tat IgG responses have also been associated with natural HIV-1 control and improved immune function in ART-treated patients receiving Tat vaccine [39][40][41][42] . Interestingly, an African study highlighted that persistent anti-Tat IgM in addition to IgG might be protective against disease progression 41 .
An IgG2 antibody response to Gag has been associated with CD4+ T cell response and long-term nonprogression 43 . The child had both responses. The weak Gag-specific CD4+ T cell response without a detectable CD8+ T cell response is intriguing. Such responses are reported in HIV-exposed uninfected individuals and some HIV-uninfected individuals, only when using sensitive cultured ELISpot assays 44 . However, the opposite exists in untreated HIV-1-infected infants where we readily demonstrated Gag-specific CD8+, but not CD4+ HIV-specific T cell responses in the first few months of life-using the same whole blood intracellular cytokine assay as for the present study 45 . Furthermore, there is an absence of sustained HIV-specific T cell responses in early ART-treated HIV-1-infected children 46 . The CD4+ Gag response demonstrated in the child seems remarkable given early treatment and long duration of viral suppression. The CD4+ T cell response may be maintained by small amounts of virus (replication-competent or replication-defective) or Gag protein produced in vivo. Lack of CD8+ T cell response suggests that CD8+ T cells might not be currently involved in controlling levels of viraemia, supporting the possibility that HIV may be Fig. 2 Detection of HIV RNA, HIV DNA and replication-competent virus. a Viral load results at 9.5 years of age when testing the standard 1 mL of plasma (target not detected (TND); left) vs. 10 mL (middle) by the standard Roche assay. A qualitative ultrasensitive RNA nested integrase PCR (IN-qPCR) assay conducted on 3 mL plasma; DNAse treated to ensure no contaminating cell-associated HIV-1 DNA (right). b Quantitation of the total HIV-1 DNA reservoir using a semi-nested quantitative reverse transcription PCR (RT-qPCR) assay at 9.5 years. The standard curve (orange squares) shows plasmid copy number controls (1-100,000 copies) on the x-axis and corresponding cycle threshold values on the y-axis. The case replicates are shown as blue squares. Curves lower than the 10 0 (1 copy) plasmid control are counted as 1 copy. c A neighbour-joining phylogenetic tree constructed using the partial gag-PR sequence (1414 bp HXB2 nt 903-2334; Gag aa39-501, PR aa 1-28). Reference subtypes A-D (in blue, black, purple and green, respectively) are included and the tree is rooted on SIV chimpanzee sequence (Los Alamos HIV sequence database; https://www.hiv.lanl.gov). Accession numbers (e.g. AF067155) of reference sequences are indicated in the figure. Numbers at the nodes indicate percentage bootstrap scores (n = 1000). The child's (Case) gag consensus sequence (see Supplementary Fig. 1) is indicated and clusters with subtype C sequences (purple). d The ability to reactivate virus from the child's CD4+ T cells was measured using two co-culture methods: donor CD8-depleted peripheral blood mononuclear cells (PBMCs) and MOLT4/CCR5 cells (top of panel). Included is the healthy HIV-1-uninfected donor (negative control) and an HIV-1-infected patient with high viral load, CD4+ T cell count <200 cells per µL (positive control). The ability to infect CD4+ cells from the case with HIV-1 BaL (bottom of panel). Donor and MOLT4/CCR5 cells were included as positive controls. The case was tested at the indicated times (age). The different sizes and shades of blue colour of the circles represent the p24 concentration in culture supernatants; the actual pg mL defective for further infection of permissive cells. In keeping with this, we hypothesize the HIV-1 reservoir in the child may be maintained through homeostatic, antigen-stimulated or integration site-dependent proliferation of CD4+ T cells harbouring transcription/translation-competent but not necessarily replication-competent HIV-1. These cellular proliferative mechanisms present a challenge for HIV-1 eradication strategies 47 . In contrast, the French case showed weak but broader CD4+ and CD8+ T cell responses to Gag, Pol and Nef, and with readily achievable viral proliferation in culture 21 .
These collective findings raise questions of whether crossreacting antigens might, in addition to small amounts of virus production in vivo, contribute to maintaining some of these memory responses-and, if such responses contributed to remission in this child. Importantly, lack of detection does not preclude having CD8+ T cell responses 48 , or other protective responses such as ADCC/phagocytosis in the presence of maternal/child antibody, that may have been active early in life and possibly essential to the outcome of HIV-1 remission. The rapid decline in VL over a month prior to ART initiation may be of considerable importance in understanding the reasons behind virological control in the child. ART may have protected an early pre-ART antiviral response that may otherwise have been compromised by continued viral replication.
The child had no HLA class I alleles shown in adults to associate with HIV-1 control 49 , except for heterozygosity at all HLA loci that is considered advantageous 50 . In contrast, the French case was homozygous at three loci, considered disadvantageous 21 . The child possesses HLA class II alleles already associated with the robust mucosal CD4+ T cell responses in elite   Fig. 3 HIV-specific responses and immune response capability of the case at 9.5 years of age. a Detection of HIV-specific antibodies at 9.5 years of age by western blot. The case antibody profile is compared with controls that are a high positive, low positive and HIV-negative. HIV proteins corresponding to bands in the blots are shown in the left grey-shaded block; the case profile was positive for the core proteins indicated in pink. b Quantitation of HIVspecific antibodies by multiplex bead array for all isotypes and subclasses (indicated on the left side-IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2) against gp41, Gag, RT, gp120, Nef, Tat, Vpu, peptide Vpu9 and V1V2 scaffold antigens (indicated at the top). Results are expressed as mean fluorescence intensities (MFI)), the colour key shows ranges of MFI according to colour intensity (the darker the more HIV-specific antibody detected). A result is considered positive if above the cut-off (mean ± 3 SD) determined from eight adult uninfected controls. Vpu9 amino acid sequence: STMVDMGHLRLLDVNDL. c Proportions of natural killer (NK) cells that respond to anti-CD16 antibody, and CD4+ and CD8+ T cells that respond to staphylococcal enterotoxin B (SEB) in a whole blood intracellular cytokine (ICC) assay that measures induction of interferon-γ (IFN-γ) and interleukin-2 (IL-2). HIV-uninfected adult reference values for comparison (n = 21; median % and range)-natural killer (NK) anti-CD16%: 37.92 (12-67.6), CD4 SEB%: 6.04 (0.25-11.91), CD8 SEB%: 5.82 (0. 18-18.94). d A weak positive CD4+ T cell response to Gag (0.116%) in the absence of a detectable CD8+ T cell response to Gag (<0.1%; 0.023%). UN: addition of costimulatory antibodies anti-CD28 and anti-CD49d, no stimulation with peptides controllers (DQB1*06 and DRB1*13:02:01) 51 . DQB1*06 has also been associated with IgA responses to V1V2 and increased risk of acquisition in the RV144 HIV vaccine trial 52 . Possession of these class II alleles may explain why the CD4+ Gag response remains detectable at 9.5 years, despite the low-level antigenic exposure. KIR genotyping revealed some features already associated with greater risk of vertical transmission in mother-to-child HIV transmission studies 24,25 .
The child had a healthy CD4:CD8 T cell ratio and levels of immune activation similar to healthy uninfected children of similar age. Robust T cell and NK cell responses to stimuli suggested a good immune response capability. CCR5 density on T cells was amongst the lowest when compared to HIVuninfected children or adults-a feature that may be advantageous. Although the child's PD-1-expressing CD4+ T cell proportions were higher than children and adults, PD-1-expressing CD8+ T cells were comparable to adults but much higher than children. High PD-1 expression is unlikely from immune exhaustion given no evidence of high immune activation in the child unlike in chronic untreated and virologically suppressed ART-treated HIV-1 infection 53,54 . In this regard TIGIT, another marker of immune exhaustion, is not different from that in uninfected children. Overall, these features show an immune system that closely resembles that of an uninfected child of similar age, making this child an ideal example of post-treatment HIV-1 control.
The ability of this child to both control virus to levels below detection on standard assays and to control immune activation presents the best of both worlds; these features have been described separately for elite controllers 18,55 , non-pathogenic nonhuman primate Simian immunodeficiency virus infection and long-term non-progressing children 56,57 . Events that may have led to sufficient silencing of virus replication will be explored by whole-genome sequencing of virus/provirus. Further investigation will expand our understanding of how the immune system controls HIV-1 replication with accompanying low immune activation, and inform future strategies for ART interruption and other interventions for HIV remission.

Methods
The case and study participants. In the CHER trial (ClinicalTrials.gov Identifier: NCT00102960; 2005-2011), HIV-1-infected treatment-naive, asymptomatic infants aged ≤12 weeks with a CD4% ≥25% were randomized to early limited ART for 40 (ART-40W; n = 143) or 96 (ART-96W; n = 143) weeks or to deferred ART (n = 125) 23 . In the ART-40W group, 122 met criteria to stop ART, together with 105 in the ART-96W group. Re-initiation of ART was based on a CD4% decrease to <20% and on clinical criteria 23 . By the trial end, median follow-up was 4.9 years (IQR: 3.7-5.3). The child in this report was randomized to the ART-40W arm.
Follow-up after the trial continued as part of PEPFAR-supported routine health services and later in an observational study 58,59 . CD4+ T cell counts were determined using the FACSCount System (BD Biosciences, San Jose, CA, USA).
Plasma HIV-1 RNA levels were quantitated retrospectively on stored samples by Roche Ampliprep/COBAS ® Taqman HIV-1 Test, v2.0 (Roche Molecular Systems, Inc., Branchburg, NJ, USA) with a lower detection limit of 20 RNA copies per mL (except for three time points where detection limits of assays used were 50 or 400 RNA copies per mL).
To gain further insight into specific characteristics of this case, detailed virological, immunological and genetic studies were undertaken at 9.5 years of age. Some viral studies were conducted on stored samples from 50 weeks of age at ART interruption. The mother is deceased and no stored maternal samples are available for study.
Ethics statement. The CHER trial was approved by the Ethics Committees of the University of the Witwatersrand and Stellenbosch University. Thereafter, the Human Research Ethics Committee of the University of the Witwatersrand provided approval for all subsequent observational studies and investigations of the Case. All participants provided written informed consent. For all minors, a parent or guardian gave written informed consent and the child gave written assent. We have complied with all ethical regulations.
Assays to detect HIV-1 DNA and RNA. An sn-qPCR assay targeting reverse transcriptase (RT) was developed for subtype C proviral DNA quantitation based on methods of Pasternak et al. 60 and Kiselinova et al 61 , and described in Kuhn et al 28 .
The sn-RT-qPCR is a two-step PCR in which the first round of amplification was carried out using conventional PCR and allowed to proceed for 15 cycles only and the total product of the first-round PCR was subsequently used in the second round of PCR, a real-time hydrolysis probe-based PCR using a fluorescently labelled TaqMan probe, one primer identical to the forward primer used in the first-round PCR and a second reverse primer designed to bind "deeper" within the amplicon. The second-round PCR was carried out for the standard cycle numbers used in realtime PCR (40 cycles). The numbers of HIV-1 proviral DNA copies were determined by using the standard curve method. To construct the standard curve, known copies of linearized HIV-1 p8.MJ4 plasmid DNA were serially diluted and amplified in a background of HIV-1-negative human gDNA (the same amount used in the experimental wells) and subjected to the same cycling conditions. Sequences of primers and probe are as follows: first-round PCR, sn-RT-forward primer-1 5′-CAT TTC TTT GGA TGG GGT ATG A-3′ and sn-RT-reverse primer-1 5′-CCT GTT CTC TGC CAA TTC TAA TTC TGC-3′; second-round qPCR, forward primer identical sn-RT-forward primer-1 above and sn-RT-reverse primer-2; 5′-TTG CCC AGT TTA ATT TTC CCA CTA-3′; sn-RT-probe; 5′−6 FAM-AGC TGG ACT GTC AAT GA-MGB-3′. gDNA was extracted using the QIAamp DNA Blood kit (Qiagen, Hilden, Germany). Conventional PCR was performed using the Roche Expand High-Fidelity (HiFi) PCR System (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany) and the qPCR was carried out using LightCycler 480 Probes Master Mix (Roche Diagnostics). Cycling conditions were standard for both Expand HiFi and TaqMan hydrolysis probe qPCR. At 9.5 years of age, a total of 9 µg of gDNA extracted from isolated PBMCs (cell equivalent is 1.36 × 10 6 cells) was tested at an input of 1 µg per well. The standard curves were run in triplicate at each plasmid dilution, except for the 1 copy standard where five replicates were done. At 50 weeks of age, 3 µg of PBMC DNA (cell equivalent is 4.54 × 10 5 cells) was tested.  An in-house ultrasensitive n-qPCR, targeting the highly conserved integrase (IN) gene designed using all subtype C sequences available on the Los Alamos HIV Database (https://www.hiv.lanl.gov/), was used to detect subtype C HIV-1 DNA and RNA. Briefly, the n-qPCR assay is a two-step PCR with the first PCR being a standard PCR allowed to proceed to end-point (30 cycles) using the Roche Expand HiFi PCR system and the second PCR a hydrolysis probe real-time PCR using Roche LightCycler 480 Probes Master Mix. The total product generated in the first-round PCR is used in the second round. Negative controls include both water and gDNA or . c CCR5 density and percentages of cells expressing CCR5, HLA-DR (immune activation), TIGIT and PD-1 (immune checkpoint inhibitory molecules) on CD4+ and CD8+ T cells. Uninfected children were compared to adults using two-tailed non-parametric Mann-Whitney U tests; individual data points are shown as coloured circles; medians are shown as solid lines; statistical significance at P < 0.05 is indicated. One adult outlier (extreme) data point was omitted from the CD8+HLA-DR+ graph; the adult vs. children comparison was not statistically different with or without this data point (P > 0.05) ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08311-0 extracted from plasma or culture supernatant using the Qiagen QIAamp Viral RNA kit. Complementary DNA was synthesized using the Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific, MA, USA) and a combination of a gene-specific primer (reverse primer-1) and random hexamers. DNase treatment of extracted RNA was carried out using the Ambion TURBO DNAfree kit (Thermo Fisher Scientific). Cycling conditions were standard for both Expand HiFi and TaqMan hydrolysis probe qPCR.
HIV-1 proviral gag sequencing. The near full-length HIV-1 gag gene encompassing partial p17 Gag coding regions to the Gag stop codon was PCR-amplified from gDNA isolated from CD4+ T cells using an in-house nested PCR assay. A first-round PCR amplification was performed using outer PCR primers gagoutF (5′-TGT TAA AAC ACT TAG TAT GGG CAA G-3′) and gagoutR (5′-TTA CTT TGA TAA AAC CTC CAA TTC C-3′) using the Roche FastStart HiFi PCR system (Roche Diagnostics). A second-round PCR amplification was performed using a tenth volume of first-round PCR product and second-round PCR primers gaginF Tween-20, 0.05% sodium azide), counted using a haemocytometer and stored in the dark at 4°C. HIV-specific immunoglobulin isotypes were detected by preparing a microsphere mixture comprising the seven HIV antigen-coupled beads (gp41, Gag, RT, gp120, Nef, Tat, Vpu, peptide Vpu9 and V1V2 scaffold; Supplementary Table 4). To each well of a 96-well flat bottom Greiner plate (Bio-Rad), 50 µL of working microsphere mixture prepared in assay buffer (PBS, 1% BSA; 2500 beads from each bead region per well), 40 µL assay buffer and 25 µL of patient plasma (diluted 1:75 in assay buffer) was added. Following an incubation period (on a shaker in the dark at room temperature; 2 h), the plate was washed three times with wash buffer (PBS, 0.1% BSA, 0.05% Tween-20) with a final wash in assay buffer. HIV-specific antibody isotypes were detected by adding 50 µL per well at 2 µg mL −1 , R-phycoerthyrinconjugated mouse anti-human IgG 1 to IgG 4 (Cat. Nos 9052-09, 9070-09, 9210-09, 9200-09, respectively, Southern Biotech, USA), mouse anti-human IgM (Cat. No. 9020-09, Southern Biotech), mouse anti-human IgA 1 (Cat. No. 9130-09, Southern Biotech) or mouse anti-human IgA 2 (Cat. No. 9140-09, Southern Biotech) with shaking (in the dark at room temperature) followed by three washes. Beads were finally resuspended in Bio-Rad sheath fluid and analysed using the Bio-Plex 200 instrument (Bio-Rad) by measuring the fluorescence signal for 50 beads per analyte. Background signal, defined as the mean fluorescence intensity (MFI) for each microsphere set when incubated with PE detection Ab in the absence of patient plasma, is subtracted from the fluorescent intensity of each sample. The cut-off for seropositivity for each analyte is calculated as the average MFI value from pooled plasma from HIV-1-uninfected patients plus 3 standard deviations. HIV-Ig (Cat. No. 3957; NIH AIDS Reagent Programme) served as a positive control on each plate and the MFI values tracked with a Levy-Jennings plot.
Host genotyping. HLA class I and II and KIR genotyping was conducted on gDNA extracted from blood cells (QiaAmp DNA blood mini kit, Qiagen). The sequencebased typing resolver kits (Conexio Genomics, Fremantle, Australia) were used to generate HLA-A, HLA-B, HLA-C, DRB1, DPB1 and DQB1 amplicons for DNA sequencing as described by the manufacturers. The exons included for DNA sequencing are: exons 1-4 for HLA-A and HLA-B; exons 1-8 for HLA-C; exons 2 and 3 for HLA-DRB1 and HLA-DQB1; exons 1-5 for HLA-DPB1.
The presence or absence of the 16 KIR genes (14 functional and 2 pseudogenes 2DP1 and 3DP1) were determined using allele-specific (AS) primers (Supplementary Table 5) in a real-time PCR assay 67 . Briefly, PCR reactions were performed in a 5 µL volume, containing 2× Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Burlington, ON, Canada), 0.2 µM of KIR-specific primers, 0.2 µM of galactosylceramidase-specific primers and 5 ng of DNA. Thermocycling was performed using the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95°C for 10 min, followed by 30 cycles of 95°C for 15 s and 60°C for 1 min, with subsequent melt-curve analysis.
Published primers and probe specific for KIR2DS4 full-length (f; *001) or truncated (v; *003,004,006,007,009) alleles 68 and KIR3DL1/S1 69 were used in combination with the primers and probe specific for the human β-globin (BGB) reference gene 70 (Supplementary Table 6) in a probe hydrolysis-based relative quantification real-time allele-specific (AS)-PCR assay to determine gene copy number variation (KIR2DS4 and KIR3DL1/3DS1). To facilitate target and reference gene multiplexing, KIR-specific probes were labelled at the 5′ end with the fluorochrome VIC, while BGB probes were labelled with the fluorochrome FAM. Control samples of known gene copy numbers were run concurrently with unknowns and gene copy numbers inferred using a delta Ct method (difference between Ct values obtained for KIR and BGB genes). All samples were run in duplicate. Real-time AS-PCR amplification was performed in 96-microwell PCR plates using an ABI7500 real-time PCR instrument (Life Technologies, Carlsbad, CA, USA). Reaction volumes were 5 µL containing 5 ng of genomic DNA, 2× LightCycler 480 Probes Master Mix (Roche), 0.5 µM KIR3DL1/S1 or KIR2DS4f/v forward/reverse primers (Inqaba Biotec, Pretoria, RSA), 0.5 µM BGB forward/ reverse primers (Inqaba Biotec, Pretoria, RSA), 0.1 µM VIC-labelled KIR3DL1/S1 or KIR2DS4f/v probe and 0.1 µM FAM-labelled BGB probe (Life Technologies, Carlsbad, CA, USA). Cycling conditions were an initial incubation of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 55°C for 10 s and 60°C for 30 s.