Routine HIV testing of pregnant women was conducted to confirm HIV status in accordance with the Western Cape prevention of mother-to-child transmission (PMTCT) HIV guidelines (Pellowski et al., 2019). All HIV-exposed children received HIV testing as per local guidelines, and all HEU children in the sub-sample were confirmed negative. Detection of HIV was done at 6 weeks by PCR and at 9 months and 18 months by rapid antibody, PCR or ELISA (Zar et al., 2015).

Maternal CD4 cell count and viral load during pregnancy were measured, with the result closest to 26 weeks’ gestation taken to coincide with the immune variables. Viral load was categorized as below the detectable limit with < 40 copies/ml, detectable with 40–1000 copies/ml and unsuppressed with > 1000 copies/ml. All mothers living with HIV received antiretroviral therapy (ART) during pregnancy according to PMTCT guidelines at the time. Maternal ART initiation was categorized as ‘before pregnancy’ or ‘during pregnancy’. All HEU infants received prophylaxis (nevirapine alone or combined with zidovudine) from birth (Pellowski et al., 2019).

Blood serum samples were taken at 26 weeks gestation for mothers and at 6–10 weeks and 24–28 months for children as outlined in the DCHS (Zar et al., 2015).

Sociodemographic information was collected using an interviewer administered questionnaire adapted from items used in the South African Stress and Health Study. Mothers self-reported employment status, education level, asset ownership, household income and clinic during an antenatal study visit between 28- and 32-weeks gestation (Stein et al., 2015, Zar et al., 2015).

Upon delivery detailed birth data was obtained, including mode of delivery, gestational age, infant sex, head circumference, infant length and infant birth weight. Gestational age was calculated using the best estimated delivery date based on the last menstrual period, antenatal ultrasound, or the symphysis-fundal height. Prematurity was defined at <37 weeks gestation (Budree et al., 2017b). Maternal Body Mass Index (BMI) was determined at 6 weeks postpartum (Budree et al., 2017b). Self-reported information on child feeding practices was obtained at infant follow-up visits at 6–10 weeks, and 24–28 months of age. At 6–10 weeks infants were categorized as exclusively breastfed if mothers were still breastfeeding but neither solids nor formula had been introduced (Budree et al., 2017a, Wedderburn et al., 2019b, Zar et al., 2015).

Maternal alcohol use during pregnancy was assessed using the Alcohol, Smoking, and Substance Involvement Screening Test; mothers were classified with either moderate-severe alcohol exposure vs. un-exposed (Stein et al., 2015). Infants and children were classified as being alcohol-exposed in utero if their mother was classified with moderate-severe alcohol exposure. Smoke exposure was measured at 26 weeks gestation using urine cotinine levels that were determined using the IMMULITE 1000 nicotine metabolite kit (Vanker et al., 2016). Mothers were considered a non-smoker if cotinine levels were < 10 ng/ml, a passive smoker with levels between 10 and 500 ng/ml and an active smoker with levels > 500 ng/ml.

Neurodevelopment of the children at 24–28 months was assessed with the Bayley Scales of Infant and Toddler Development, third edition (BSID-III) assessment (Bayley, 2006, Donald et al., 2018). The BSID-III is a well-validated tool that assesses child cognitive, language and motor development from 1 to 42 months and is sensitive to developmental delay (Bayley, 2006). The BSID-III testing was performed by trained assessors that were blinded to maternal HIV status. Training was performed in accordance with the BSID manual by a pediatric neurodevelopmental specialist who periodically monitored the assessors throughout the testing period to validate standardized data collection across sites and ensure agreement between assessors on both administration and scoring. The assessors alternated between the TC Newman and Mbekweni clinics and assessed equal number of children at each site. Assessments were performed with prompts in the child’s preferred language. External scoring quality control checks were also performed centrally before data capture. For the current study standardized composite scores were used. Composite scores were generated for each cognitive, language and motor domain, with a mean of 100 and standard deviation of 15, using normative United States data. The use of these composite scores have been validated in the South African setting (Ballot et al., 2017, Rademeyer and Jacklin, 2013).

Pro- and anti-inflammatory cytokines have been used as an indicator of immune regulation in the majority of existing studies with HEU children (Borges-Almeida et al., 2011, Chougnet et al., 2000, Dirajlal-Fargo et al., 2019, Miyamoto et al., 2017, Prendergast et al., 2017), pregnant women living with HIV (Faye et al., 2007, Maharaj et al., 2017, Moussa et al., 2001, Richardson and Weinberg, 2011, Sachdeva et al., 2008) and brain disorders in children (Miyamoto et al., 2017). Serum neutrophil gelatinase-associated lipocalin (NGAL) levels were shown to be associated with cognitive impairments and reduced brain volumes in people living with HIV (Williams et al., 2019, Williams et al., 2020). NGAL levels were increased in the neocortex of post-mortem brain tissues from a subset of people living with HIV that had HIV-associated neuropathology. NGAL levels correlated with viral load in the CSF and pro-viral DNA in the cortex (Ojeda-Juárez et al., 2020). The study by Ojeda-Juárez and colleagues further showed that NGAL may play an important function in neuronal damage and neuroinflammation in a HIVgp120 transgenic mouse model for HIV (Ojeda-Juárez et al., 2020). Decreased plasma MMP-9 levels were found in South African people living with HIV (Williams et al., 2019). Evidence from studies in animals show that optimal maternal and infant MMP-9 levels are crucial for early life brain developmental processes (Reinhard et al., 2015).

NGAL and MMP-9 concentrations were measured using commercially available ELISA kits (NGAL: DY1757, MMP-9: D911; R&D systems) in serum samples obtained from the mothers at 26 weeks gestation and the HEU children at 6–10 weeks and 24–28 months. Pro- and anti-inflammatory immune markers (GM-CSF, INF-γ, IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8 and TNF-α, IL-4, IL-10, IL-12p70 and IL-13) were analyzed with a Milliplex® Luminex premix 13-plex kit (HSTCMAG28SPMX13; Merck) according to the manufacturer’s instructions. Plates were read on a Luminex system (Bio-Plex 200 System; Bio-Rad). All samples were assayed in duplicate.

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