Immune Modulation of HIV-1 Reservoir Size in Early-Treated Neonates.

Immune mechanisms that modulate human immunodeficiency virus-1 (HIV-1) reservoir size in neonates are poorly understood. Using samples from neonates who initiated antiretroviral therapy shortly after birth, we demonstrate that interleukin-8-secreting CD4 T cells, which are selectively expanded in early infancy, are more resistant to HIV-1 infection and inversely correlated with the frequency of intact proviruses at birth. Moreover, newborns with HIV-1 infection displayed a distinct B-cell profile at birth, with reduction of memory B cells and expansion of plasmablasts and transitional B cells; however, B-cell immune perturbations were unrelated to HIV-1 reservoir size and normalized after initiation of antiretroviral therapy. Clinical Trials Registration. NCT02369406.

Safety and Efficacy of Starting Antiretroviral Therapy in the First Week of Life.

BACKGROUND: Early antiretroviral therapy (ART) is recommended for infants with human immunodeficiency virus (HIV) infection. However, few antiretroviral options are available for neonates. METHODS: The Early Infant Treatment Study in Botswana tested HIV-exposed infants within 96 hours of birth, and HIV-infected infants started nevirapine (NVP) 6 mg/kg twice daily, zidovudine (ZDV), and lamivudine (3TC) at age < 7 days. NVP trough concentrations were tested at 1 and 2 weeks. NVP was switched to ritonavir-boosted lopinavir (LPV/r) at week 2, 3, 4, or 5 according to delivery gestational age. RESULTS: Forty HIV-infected infants started ART at median age 2 days (range, 1-5 days). NVP trough concentrations were highly variable and below therapeutic target (3000 ng/mL) for 50% of 2-week measurements; concentrations did not correlate with viral decline at weeks 2, 4, or 12. Two deaths unrelated to ART occurred through 24 weeks. Only 1 unscheduled treatment modification was required. Within 4 weeks of transition to LPV/r, 9 (22.5%) had transient HIV RNA increases, likely due to poor LPV/r palatability. At 12 weeks, 22 (55%) of 40 were <40 copies/mL (93% <400 copies/mL); by 24 weeks, 27 of 38 (71%) were < 40 copies/mL (84% < 400 copies/mL). HIV-1 RNA response at 12 and 24 weeks did not differ by baseline HIV RNA or other factors. CONCLUSIONS: NVP/ZDV/3TC started in the first week of life was safe and effective, even when trough NVP levels were below target. Transient viral increases occurred following transition to LPV/r, but by 12 and 24 weeks most children achieved and maintained viral suppression. CLINICAL TRIALS REGISTRATION: NCT02369406.

Viral Reservoir in Early-Treated Human Immunodeficiency Virus-Infected Children and Markers for Sustained Viral Suppression.

BACKGROUND: The impact of very early infant treatment on human immunodeficiency virus (HIV) reservoir, and markers for treatment success, require study. METHODS: The Early Infant Treatment Study (EIT) enrolled 40 children living with HIV started on antiretroviral treatment (ART) at <7 days of age, with 23 who had started treatment between 30-365 days to serve as controls. Quantitative HIV DNA was evaluated every 1-3 months in peripheral blood mononuclear cells. 84-week repeat qualitative whole blood DNA polymerase chain reaction and dual enzyme immunosorbent assay were performed. RESULTS: Median quantitative cell-associated DNA after at least 84 weeks was significantly lower among the first 27 EIT children tested than among 10 controls (40.8 vs 981.4 copies/million cells; P < .001) and correlated with pre-ART DNA. Median DNA after 84 weeks did not differ significantly by negative or positive serostatus at 84 weeks (P = .94), and appeared unaffected by periods of unsuppressed plasma RNA from 24-84 weeks (P = .70). However, negative 84-week serostatus was 67% predictive for sustained RNA suppression, and positive serostatus was 100% predictive for viremia. Loss of qualitative DNA positivity at 84 weeks was 73% predictive for sustained suppression, and persistent positivity was 77% predictive for viremia. CONCLUSIONS: Lower viral reservoir was associated with starting ART at <1 week. Negative serostatus and qualitative DNA were useful markers of sustained viral suppression from 24-84 weeks.

Caregivers of children with HIV in Botswana prefer monthly IV Broadly Neutralizing Antibodies (bNAbs) to daily oral ART.

INTRODUCTION: Monthly intravenous infusion of broadly neutralizing monoclonal antibodies may be an attractive alternative to daily oral antiretroviral treatment for children living with HIV. However, acceptability among caregivers remains unknown. METHODS: We evaluated monthly infusion of dual bNAbs (VRCO1LS and 10-1074) as a treatment alternative to ART among children participating in the Tatelo Study in Botswana. Eligible children aged 2-5 years received 8-32 weeks of bNAbs overlapping with ART, and up to 24 weeks of bNAbs alone as monthly intravenous infusion. Using closed-ended questionnaires, we evaluated caregiver acceptability of each treatment strategy prior to the first bNAb administration visit (pre-intervention) and after the completion of the final bNAb administration visit (post-intervention). RESULTS: Twenty-five children completed the intervention phase of the study, and acceptability data were available from 24 caregivers at both time points. Responses were provided by the child's mother at both visits (60%), an extended family member at both visits (28%), or a combination of mother and an extended family member (12%). Caregiver acceptance of monthly bNAb infusions was extremely high both pre-and post-intervention, with 21/24 (87.5%) preferring bNAbs to ART pre-intervention, and 21/25 (84%) preferring bNAbs post-intervention. While no caregiver preferred ART pre-intervention, 2/25 preferred it post-intervention. Pre-intervention, 3 (13%) caregivers had no preference between monthly bNAbs or daily ART, and 2 (8%) had no preference post-intervention. Pre-intervention, the most common reasons for preferring bNAbs over ART were the perception that bNAbs were better at suppressing the virus than ART (n = 10) and the fact that infusions were dosed once monthly compared to daily ART (n = 9). Post-intervention, no dominant reason for preferring bNAbs over ART emerged from caregivers. CONCLUSIONS: Monthly intravenous bNAb infusions were highly acceptable to caregivers of children with HIV in Botswana and preferred over standard ART by the majority of caregivers. CLINICAL TRIAL NUMBER: NCT03707977.

Brief Report: Long-Term Clinical, Immunologic, and Virologic Outcomes Among Early-Treated Children With HIV in Botswana: A Nonrandomized Controlled Clinical Trial.

BACKGROUND: Early antiretroviral treatment (ART) improves outcomes in children, but few studies have comprehensively evaluated the impact of ART started from the first week of life. METHODS: Children diagnosed with HIV within 96 hours of life were enrolled into the Early Infant Treatment Study in Botswana and followed on ART for 96 weeks. Nevirapine, zidovudine, and lamivudine were initiated; nevirapine was switched to lopinavir/ritonavir between weeks 2-5 in accordance with gestational age. Clinical and laboratory evaluations occurred at weeks 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, and 96. FINDINGS: Forty children initiated ART at a median of 2 (IQR 2, 3) days of life; 38 (95%) completed follow-up through 96 weeks, and 2 (5%) died between 12 and 24 weeks. ART was well tolerated; 9 children (24%) experienced a grade 3 or 4 hematologic event, and 2 (5%) required treatment modification for anemia. The median 96-week CD4 count was 1625 (IQR 1179, 2493) cells/mm 3 with only 5/38 (13%) having absolute counts <1000 cells/mm 3 . Although 23 (61%) had at least one visit with HIV-1 RNA ≥40 copies/mL at or after 24 weeks, 28 (74%) had HIV-1 RNA <40 copies/mL at the 96-week visit. Median cell-associated HIV-1 DNA at 84/96-week PBMCs was 1.9 (IQR 1.0, 2.6) log 10 copies/10 6 cells. Pre-ART reservoir size at birth was predictive of the viral reservoir at 84/96 weeks. INTERPRETATION: Initiation of ART in the first week of life led to favorable clinical outcomes, preserved CD4 cell counts, and low viral reservoir through 96 weeks of life.

Broadly neutralizing antibody treatment maintained HIV suppression in children with favorable reservoir characteristics in Botswana.

Broadly neutralizing antibodies (bNAbs) may provide an alternative to standard antiretroviral treatment (ART) for controlling HIV-1 replication and may have immunotherapeutic effects against HIV-1 reservoirs. We conducted a prospective clinical trial with two HIV-1 bNAbs (VRC01LS and 10-1074) in children (n = 25) who had previously initiated small-molecule ART treatment before 7 days of age and who continued treatment for at least 96 weeks. Both bNAbs were dosed intravenously every 4 weeks, overlapping with ART for at least 8 weeks and then continued for up to 24 weeks or until detectable viremia of HIV-1 RNA rose above 400 copies per milliliter in the absence of ART. Eleven (44%) children maintained HIV-1 RNA below 400 copies per milliliter through 24 weeks of bNAb-only treatment; 14 (56%) had detectable viremia above 400 copies per milliliter at a median of 4 weeks. Archived HIV-1 provirus susceptible to 10-1074, lower birth HIV-1 DNA reservoir in peripheral blood mononuclear cells, sustained viral suppression throughout early life, and combined negative qualitative HIV-1 DNA polymerase chain reaction and negative HIV-1 serology at entry were associated with maintaining suppression on bNAbs alone. This proof-of-concept study suggests that bNAbs may represent a promising treatment modality for infants and children living with HIV-1. Future studies using newer bNAb combinations with greater breadth and potency are warranted.

Immune correlates of HIV-1 reservoir cell decline in early-treated infants.

Initiation of antiretroviral therapy (ART) in infected neonates within hours after birth limits viral reservoir seeding but does not prevent long-term HIV-1 persistence. Here, we report parallel assessments of HIV-1 reservoir cells and innate antiviral immune responses in a unique cohort of 37 infected neonates from Botswana who started ART extremely early, frequently within hours after birth. Decline of genome-intact HIV-1 proviruses occurs rapidly after initiation of ART and is associated with an increase in natural killer (NK) cell populations expressing the cytotoxicity marker CD57 and with a decrease in NK cell subsets expressing the inhibitory marker NKG2A. Immune perturbations in innate lymphoid cells, myeloid dendritic cells, and monocytes detected at birth normalize after rapid institution of antiretroviral therapy but do not notably influence HIV-1 reservoir cell dynamics. These results suggest that HIV-1 reservoir cell seeding and evolution in early-treated neonates is markedly influenced by antiviral NK cell immune responses.

Safety and Pharmacokinetics of Intravenous 10-1074 and VRC01LS in Young Children.

BACKGROUND: Broadly neutralizing monoclonal antibodies (bNAbs) suppress HIV-1 RNA and may deplete residual viral reservoirs. We evaluated the safety and pharmacokinetics (PK) of dual intravenous VRC01LS and 10-1074 in very early-treated children with HIV-1 on suppressive antiretroviral treatment (ART). SETTING: Botswana. METHODS: Children with HIV-1 (median age 3.1 years) on ART from <7 days old were enrolled. In phase A, 6 children received 10-1074 (30 mg/kg at day 0, 28, and 56) and 6 children received VRC01LS (30 mg/kg at day 0, 10 mg/kg at days 28 and 56) by intravenous infusion. In phase B, 6 children received the 2 bNAbs combined (with higher VRC01LS maintenance dose, 15 mg/kg) every 4 weeks for 32 weeks with PK evaluations over 8 weeks. Population PK models were developed to predict steady-state concentrations. RESULTS: BNAb infusions were well tolerated. There were no infusion reactions nor any bNAb-related grade 3 or 4 events. The median (range) first dose Cmax and trough (day 28) combined from both phases were 1405 (876-1999) µg/mL and 133 (84-319) µg/mL for 10-1074 and 776 (559-846) µg/mL and 230 (158-294) µg/mL for VRC01LS. No large differences in bNAb clearances were observed when given in combination. The estimated VRC01LS half-life was shorter than in adults. Predicted steady-state troughs [median (90% prediction interval)] were 261 (95-565) and 266 (191-366) µg/mL for 10-1074 and VRC01LS, respectively, when given in combination. CONCLUSIONS: 10-1074 and VRC01LS were safe and well-tolerated among children receiving ART. Troughs exceeded minimal targets with every 4-week administration of 10-1074 at 30 mg/kg and VRC01LS at 15 mg/kg.

Patterns of pretreatment drug resistance mutations of very early diagnosed and treated infants in Botswana.

OBJECTIVE: To describe the occurrence of HIV drug resistance mutations (DRMs) in both intact and defective HIV-1 cell-associated DNA (HIV-1 CAD) among early-treated infants. DESIGN: The Botswana EIT Study (ClinicalTrials.gov NCT02369406) initiated antiretroviral therapy (ART) in the first week of life and evaluated HIV-1 in plasma and peripheral blood mononuclear cells (PBMCs). METHODOLOGY: We analyzed 257 near-HIV-1 full-length sequences (nFLS) obtained by Illumina next-generation sequencing from infants near birth. Sanger sequencing of pol was performed for mothers at delivery and children with clinical failure through 96 weeks. DRMs were identified using the Stanford HIV Drug Resistance Database. RESULTS: In 27 infants, median PBMC HIV-1 proviral load was 492 copies/ml [IQR: 78-1246 copies/ml] at a median of 2 days (range 1-32); 18 (66.7%) had no DRMs detected; six (22.2%) had DRMs detected in defective DNA only, and three (11.1%) had DRMs in both defective and intact DNA (P = 0.09). A total of 60 of 151 (37.7%) defective sequences had at least one DRM: 31.8% NNRTI, 15.2% NRTI, 5.3% protease inhibitor, and 15.5% INSTI-associated mutations. In intact sequences, 33 of 106 (31.1%) had at least 1 DRM: 29.2% NNRTI, 7.5% NRTI, 0.9% protease inhibitor, and no INSTI-associated mutations. For all three infants with intact sequence DRMs, corresponding DRMs occurred in maternal plasma at delivery. Archived DRMs were detectable at a later clinical rebound on only one occasion. CONCLUSION: Defective HIV-1 cell-associated DNA sequences may overestimate the prevalence of drug resistance among early-treated children. The impact of DRMs from intact proviruses on long-term treatment outcomes warrants further investigation.

HIV diagnostic algorithm requires confirmatory testing for initial indeterminate or positive screens in the first week of life.

BACKGROUND: Risk for nondiagnostic and false-positive HIV testing has not been quantified for neonates. METHODS: From April 2015 to July 2018, we screened HIV-exposed infants in Botswana less than 96 h from birth by qualitative DNA PCR. Repeat blood draws for DNA and RNA PCR testing occurred for initial positive and indeterminate results to establish final diagnosis. We compared screening DNA PCR cycle threshold values with final HIV status of the child. RESULTS: Of 10 622 HIV-exposed infants, 10 549 (99.3%) had no HIV DNA detected (negative), 42 (0.4%) had HIV DNA detected (positive), and 31 (0.3%) tested indeterminate at first HIV screen. Repeat testing identified 2 (5.0%) of 40 positive screens (2 declined additional testing) as false positives and confirmed 2 (6.5%) of 31 indeterminate screens as infected. Median cycle threshold value at screening was 28.1 (IQR 19.8--34.8) for children with final positive status, and 35.5 (IQR 32.8--41.4) for indeterminates who were ultimately negative. Six (15%) of 40 infants with final positive status had cycle threshold value greater than 33 at first screen, whereas 3 (9.7%) of 31 indeterminates with final negative status had cycle threshold value 33 or less at first screen. This threshold resulted in a negative predictive value of 82% and a positive predictive value of 92% for a single screen. CONCLUSION: Although a DNA PCR cycle threshold value of 33 was predictive of the final HIV status in newborns, overlap occurred for true positives, false positives, and initial indeterminates. Testing additional samples should be standard practice for positive and indeterminate HIV DNA PCR tests in the first week of life.

Mother-to-Child HIV Transmission With In Utero Dolutegravir vs. Efavirenz in Botswana.

BACKGROUND: A large-scale evaluation of mother-to-child transmission (MTCT) with dolutegravir (DTG)-based antiretroviral treatment (ART) has not been conducted previously. SETTING: Botswana was the first African country to change from efavirenz (EFV)/tenofovir (TDF)/emtricitabine (FTC) to DTG/TDF/FTC first-line ART. METHODS: From April 2015 to July 2018, the Early Infant Treatment Study offered HIV DNA testing at <96 hours of life. Maternal ART regimen was available for screened infants who could be linked to the separate Tsepamo surveillance study database. We evaluated characteristics of HIV-positive infants, and compared MTCT rates by ART regimen for linked infants. RESULTS: Of 10,622 HIV-exposed infants screened, 42 (0.40%) were HIV-positive. In total, 5064 screened infants could be linked to the surveillance database, including 1235 (24.4%) exposed to DTG/TDF/FTC and 2411 (47.6%) exposed to EFV/TDF/FTC. MTCT was rare when either regimen was started before conception: 0/213 [0.00%, 95% confidence interval (CI): 0.00% to 1.72%] on DTG, 1/1497 (0.07%, 95% CI: 0.00% to 0.37%) on EFV. MTCT was similar for women starting each ART regimen in pregnancy: 8/999 (0.80%, 95% CI: 0.35% to 1.57%) for DTG and 8/883 (0.91%, 95% CI: 0.39% to 1.78%) for EFV (risk difference 0.11%, 95% CI: -0.79% to 1.06%). Most MTCT events (4/8 with DTG, 6/9 with EFV) occurred when ART was started <90 days before delivery. Infants exposed to DTG in utero had lower baseline HIV RNA compared with other HIV-infected infants. CONCLUSION: In utero MTCT in Botswana remains rare in the DTG era. No significant MTCT differences were observed between DTG/TDF/FTC and EFV/TDF/FTC. Risk was highest for both groups when ART was started in the third trimester.

Early antiretroviral therapy in neonates with HIV-1 infection restricts viral reservoir size and induces a distinct innate immune profile.

Neonatal HIV-1 infection is associated with rapidly progressive and frequently fatal immune deficiency if left untreated. Immediate institution of antiretroviral therapy (ART), ideally within hours after birth, may restrict irreversible damage to the developing neonatal immune system and possibly provide opportunities for facilitating drug-free viral control during subsequent treatment interruptions. However, the virological and immunological effects of ART initiation within hours after delivery have not been systematically investigated. We examined a unique cohort of neonates with HIV-1 infection from Botswana who started ART shortly after birth and were followed longitudinally for about 2 years in comparison to control infants started on treatment during the first year after birth. We demonstrate multiple clear benefits of rapid antiretroviral initiation, including an extremely small reservoir of intact proviral sequences, a reduction in abnormal T cell immune activation, a more polyfunctional HIV-1-specific T cell response, and an innate immune profile that displays distinct features of improved antiviral activity and is associated with intact proviral reservoir size. Together, these data offer rare insight into the evolutionary dynamics of viral reservoir establishment in neonates and provide strong empirical evidence supporting the immediate initiation of ART for neonates with HIV-1 infection.

Targeted HIV testing at birth supported by low and predictable mother-to-child transmission risk in Botswana.

INTRODUCTION: Most African countries perform infant HIV testing at 6 weeks or later. The addition of targeted testing at birth may improve retention in care, treatment outcomes and survival for HIV-infected infants. METHODS: HIV-exposed infants were screened as part of the Early Infant Treatment (EIT) study in Botswana. Screened infants were ≥35 weeks gestational age and ≥2000 g at birth. Risk factors for mother-to-child transmission (MTCT) were assessed by maternal obstetric card or verbally. Risk factors included <8 weeks ART in pregnancy, last known CD4 <250 cells/mm3 , last known HIV RNA >400 copies/mL, poor maternal ART adherence, lack of maternal zidovudine (ZDV) in labour, or lack of infant post-exposure prophylaxis. Infants underwent dried blood spot testing by Roche Cobas Ampliprep/Cobas Taqman HIV-1 qualitative PCR. RESULTS: From April 2015 to April 2016, 2303 HIV-exposed infants were tested for HIV in the EIT study. Of these, 369 (16%) were identified as high risk for HIV infection by information available at birth, and 12 (0.5% overall, 3.25% of high risk) were identified as HIV positive at birth. All 12 positive infants were identified as high risk at the time of screening, and only 2 risk factors were required to identify all positive infants: either <8 weeks of maternal ART in pregnancy (75%) or lack of maternal HIV suppression at last test (25%). CONCLUSIONS: In utero MTCT occurred only among infants identified as high risk at delivery, using information available from the mother or obstetric record. Birth testing that targets high-risk infants based on maternal ART receipt is likely to identify the majority of in utero HIV transmissions, and allows early ART initiation for these infants.

Brief Report: High Sensitivity and Specificity of the Cepheid Xpert HIV-1 Qualitative Point-of-Care Test Among Newborns in Botswana.

BACKGROUND: HIV point-of-care (POC) testing allows for early infant HIV diagnosis and treatment, but POC accuracy at birth and in the setting of antiretroviral prophylaxis for the prevention of mother-to-child HIV transmission is unknown. METHODS: We evaluated the Cepheid Xpert HIV-1 Qual POC test against the Roche Taqman HIV-1 DNA polymerase chain reaction (PCR) platform using dried blood spots from 15 HIV-infected and 75 HIV-exposed uninfected newborns. These infants were screened for HIV at <96 hours of life at 5 hospital maternity wards in Botswana; all infants received postexposure antiretroviral prophylaxis with single-dose nevirapine and zidovudine, and most mothers received 3-drug antiretroviral therapy in pregnancy and at delivery. RESULTS: Fourteen of the 15 PCR positive samples tested positive by Cepheid POC, yielding a sensitivity of 93.3% (95% confidence interval: 68.1 to 99.8). Baseline viral load among positive infants ranged from <40 to >10,000,000 copies/mL, with a median of 2403 copies/mL. The HIV RNA for the infant with false-negative POC testing was 1661 copies/mL. Of note, 2 infants with low HIV RNA (<40 and 272 copies/mL) were correctly identified as HIV positive by Cepheid POC. All the 75 PCR-negative samples tested negative by Cepheid POC, yielding a specificity of 100% (95% confidence interval: 96.1 to 100). DISCUSSION: Our study demonstrates high sensitivity and specificity for the Cepheid POC assay in the first week of life despite early infection and antiretroviral prophylaxis. This platform may be a useful approach for adding early infant HIV diagnosis to current testing programs.