Introduction

CCR5 is the major coreceptor for HIV-1 entry [1–5] and is critical for vertical transmission of HIV-1 as CCR5-tropic viruses are preferentially transmitted from mothers to their infants [6,7]. CCR5Δ32 homozygosity in infants confers protection from vertical transmission [8,9]. CCR5Δ32 heterozygosity, however, is associated with reduced risk of vertical HIV-1-infection in some studies [8,10] but not in others [11]. CCR5 expression associates with the susceptibility of cells to HIV-1 infection [12,13]. CCR5 density on the surface of CD4+ T-cells is lower in HIV-1-exposed uninfected individuals compared with HIV-1-unexposed individuals [14,15], and in HIV-1 controllers compared with noncontrollers [16,17].

HIV-1 preferentially infects activated CD4+ T-cells [18]. Thus, individuals with higher frequencies of activated CD4+ T-cells, may have increased risk of infection because of the increased availability of activated target cells. In individuals exposed to HIV-1, lower levels of immune activation are associated with a decreased risk of HIV-1 acquisition [19]. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) and programmed cell death protein-1 (PD-1 or CD279) are immune checkpoint receptors, which are upregulated upon activation of T-cells to prevent excessive immune activation [20]. Overexpression of immune checkpoint receptors occurs during HIV-1 infection and is associated with T-cell exhaustion and dysfunction [21–23]. Their expression correlates with HIV-1 disease progression [24–26] similarly to markers of immune activation [27]. Additionally, frequencies of CD8+ T-cells expressing the immune activation marker, CD38, positively correlate with frequencies of CD8+ T-cells expressing the immune checkpoint molecule, PD-1, in people with HIV-1 (PWH) [24,26]. Although immune activation and immune checkpoint molecules have contrasting functions, they correlate positively with HIV-1 disease progression and with each other. Aside from being an HIV-1 coreceptor, there is evidence supporting a role for CCR5 in T-cell activation [28,29]. Furthermore, CCR5 density on CD4+ T-cells correlates with frequencies of activated CD38+ CD8+ T-cells, independently of viral load, in PWH [30]. Although antiretroviral therapy (ART) initiation decreases cellular activation, it does not significantly impact HLA-DR- CD4+ T-cell CCR5 density [30]. CCR5 density on nonactivated CD4+ T-cells is stable over time in adults [17] and in children [31] living with HIV-1.

Given the crucial role of CCR5 in HIV-1 acquisition and disease progression, we hypothesized that CCR5 density on CD4+ T-cells associates with HIV-1 vertical transmission, and that a combined higher maternal–infant CD4+ T-cell CCR5 density would associate with a higher risk of in-utero infection. To address this, we analysed CCR5 expression (density and frequency) on CD4+ and CD8+ T-cells, as well as frequencies of CD4+ and CD8+ T-cells expressing immune activation [CCR5, inducible costimulator (ICOS) and HLA-DR] and immune checkpoint molecules (TIGIT and PD-1), in mothers and their infants in the context of risk of in-utero HIV-1 transmission.

Materials and methods Study participants

A total of 52 mother–infant dyads were enrolled into this study and were a subset of infants enrolled into the Latency and Early Neonatal Provision of Antiretroviral Drugs (LEOPARD) study at Rahima Moosa Mother and Child Hospital in Johannesburg, South Africa [32,33]. All 52 mothers were living with HIV-1. Of these, 27 transmitted HIV-1 to their infants [transmitting mothers) and 25 did not [nontransmitting mothers (NTMs)]. Infants with HIV-1 tested positive and infants without HIV-1 (HIV-exposed uninfected: HEU) tested negative on a diagnostic HIV-1 total nucleic acid (TNA) PCR on a sample collected within 48 h of birth. Enrollment samples were collected between less than 48 h and 19 days after birth (Supplemental Table 1, https://links.lww.com/QAD/D121). Samples collected between 31 August 2015 and 1 February 2017 were analysed.

This study was reviewed and approved by Human Research Ethics Committee (HREC) of the University of the Witwatersrand and the Institutional Review Board (IRB) of Columbia University. Written informed consent to participate in the study was provided by the participants’ legal guardian/next of kin.

Clinical covariates

Viral load for the mothers and infants with HIV-1 was measured using the COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0 (Roche Molecular Systems, Inc., Branchburg, USA) with a limit of detection of 20 copies/ml. CD4+ T-cell counts were measured using the TruCOUNT method (BD Biosciences, San Jose, USA). The maternal plasma viral loads and CD4+ T-cell counts were measured soon after delivery. Infant viral loads, CD4+ T-cell counts and percentages were measured at baseline before treatment.

Maternal (demographic characteristics, syphilis serology, HIV diagnosis, ART during pregnancy and breastfeeding) and infant data (mode of delivery, gestational age, birth weight, sex and antiretroviral prophylaxis) were collected.

Flow cytometry

EDTA-anticoagulated whole blood samples were stained within 6 h of collection. Briefly, 100 μl of whole blood was stained with an antibody cocktail, diluted in Brilliant Stain Buffer (BD Biosciences). Red blood cells were lysed with FACS lysing solution (BD Biosciences), samples washed and resuspended in FACSflow. Two different antibody panels were used. One panel contained CD8 PerCP (SK1), CD4 FITC (SK3), CD3 APC-H7 (SK7), PD-1 BV786 (EH12.1) and ICOS BV650 (DX29) from BD Biosciences and the other panel contained CD8 Alexa Fluor 700 (RPA-T8), CD4 BV786 (L200), CD3 APC-H7 (SK7), TIGIT APC (MBSA43), CCR5 PE (2D7) from BD Biosciences and HLA-DR PE-Cy5.5 (TU36) from Invitrogen (Waltham, USA). CCR5 density was calculated using BD QuantibriteTM beads (BD Biosciences) [34]. Samples were acquired on a four laser BD LSRFortessa X-20 (BD Biosciences) within 4 h. CS&T beads and mid-range Rainbow Fluorescent Particles (BD Biosciences) were run before sample acquisition. Compensation was performed for each experiment using BD CompBeads (BD Biosciences). Samples were analyzed using FlowJo software version 9.9.6 (BD Biosciences).

The gating strategy is shown in Supplemental Figure 1A, https://links.lww.com/QAD/D121. A representative example of expression of these markers in a mother–infant pair is shown in Supplemental Figure 1B, https://links.lww.com/QAD/D121.

Statistical analyses

Baseline clinical characteristics of the transmitting mothers vs. NTMs and the infants with HIV-1 (HIV) vs. HEU were compared using the Fisher's exact test for categorical variables and the Mann–Whitney U nonparametric test for continuous variables.

CCR5 density and the frequency of CD4+ and CD8+ T-cells expressing immune activation and immune checkpoint markers between mothers and their infants were compared using the Wilcoxon matched-pairs signed rank test. The Mann–Whitney U test was used to compare these parameters between transmitting mothers vs. NTMs and infants with HIV-1 vs. HEU infants. Spearman correlation coefficients were used to analyse correlations between maternal and infant cell subsets and markers of HIV-1 disease severity (CD4+ T-cell count, CD4+ T-cell percentage and HIV-1 viral load). The Fisher's exact test was used to compare proportions of NTM/HEU and TM/HIV dyads with high and low CCR5 density on CD4+ T-cells.

Variables associated with perinatal transmission by Mann–Whitney U test were used in logistic regression models. Univariate logistic regression analyses evaluated predictive associations between cell subsets and risk of HIV-1 transmission. Multivariate logistic regression was used to adjust for maternal viral load and CD4+ T-cell counts. Statistical analyses were performed using STATA version 12.1 (StataCorp. 2011, College Station, USA). GraphPad Prism 5 (GraphPad Software, San Diego, USA) and STATA were used for graphical presentations.

Results Characteristics of the study population

Characteristics of the mother–infant pairs are presented in Supplemental Table 1, https://links.lww.com/QAD/D121. Maternal age, education, parity, mode of delivery, breastfeeding, syphilis serology, timing of HIV-1 diagnosis, maternal CD4+ T-cell count and CD4+ : CD8+ ratio and length of gestation did not differ significantly between mothers who did and did not transmit. All mothers who did not transmit and 81% of the mothers who transmitted received ART (efavirenz, emtricitabine and tenofovir disoproxil fumarate) during pregnancy, with ART initiation occurring earlier in mothers who did not transmit. Maternal viral loads close to delivery were higher among mothers who transmitted compared with those who did not. Infant birth weight and sex did not differ between infants with and without HIV-1. All of the uninfected infants and 25 of the 27 infants with HIV-1 received nevirapine prophylaxis. Infant CD4+ : CD8+ ratio at enrollment did not differ significantly between infants with and without HIV-1.

Comparison of mothers who did or did not transmit

CCR5 density on CD4+ and CD8+ T-cells and the frequency of CD4+ and CD8+ T-cells expressing immune activation (CCR5, ICOS, and HLA-DR) and immune checkpoint receptors (TIGIT and PD-1) was compared between mothers who did and did not transmit. Mothers who transmitted had higher CCR5 density on both CD4+ and CD8+ T-cells and higher frequencies of CCR5+, ICOS+ and TIGIT+ CD8+ T-cells than mothers who did not transmit (Fig. 1).

Fig. 1:

Expression of CCR5 density, immune activation and immune checkpoint markers.

All maternal T-cell subsets that were significantly different between the two groups correlated positively with viral load and negatively with CD4+ T-cell count in the total group of mothers – with the exception of CCR5 density on CD4+ T-cells, which did not correlate with CD4+ T-cell count (Supplemental Figure 2, https://links.lww.com/QAD/D121). After adjustment for maternal viral load and CD4+ T-cell count, CCR5 density on CD4+ T-cells and the frequency of ICOS+CD8+ T-cells remained significantly associated with increased risk for transmission (Table 1 – model 1). When we evaluated which of the maternal subsets were associated with transmission taking into account the five variables together (data not shown) – CCR5 density on CD4+ and CD8+ T-cells and the frequency of ICOS+CD8+ T-cell subsets were each independently associated with transmission and remained so after adjusting for viral load and CD4+ T-cell count. These three variables were then analysed together (Table 1 – model 2), following which all were significant (unadjusted and when adjusted for viral load and CD4+ T-cell count), but in contrast to model 1, CCR5 density on CD8+ T-cells associated with reduced vertical transmission.

Table 1 - Logistic regression results predicting transmitter status from maternal subsets and acquisition of infection from infant subsets unadjusted and adjusted for maternal viral load and CD4+ T-cell counts. Unadjusted Adjusted for maternal VL Adjusted for maternal CD4+ T-cell counts Adjusted for maternal VL and CD4+ T-cell counts Subset Odds ratio 95% CI P Odds ratio 95% CI P Odds ratio 95% CI P Odds ratio 95% CI P Maternal subsets Model 1a  CCR5 density on CD4+ T-cells 1.02 1.01–1.03 0.003 1.01 1.0–1.02 0.012 1.02 1.01–1.03 0.003 1.01 1.00–1.03 0.011  CCR5 density on CD8+ T-cells 1.005 1.00–1.01 0.029 1 1.00–1.01 0.16 1.01 1.00–1.01 0.037 1.003 1.00–1.01 0.130  % CCR5+ CD8+ T-cells 1.04 1.00–1.08 0.043 1.02 0.98–1.07 0.33 1.04 1.00–1.09 0.071 1.03 0.98–1.08 0.280  % ICOS+ CD8+ T-cells 4.4 1.48–12.83 0.004 2.6 0.90–7.78 0.08 4.93 1.56–15.54 0.006 3.16 1.02–9.84 0.046  % TIGIT+ CD8+ T-cells 1.05 1.01–1.10 0.025 1.02 0.96–1.07 0.56 1.05 1.00–1.10 0.039 1.02 0.96–1.07 0.540 Model 2b  CCR5 density on CD4+ T-cells 1.06 1.02–1.10 0.006 1.06 1.02–1.11 0.009 1.06 1.01–1.10 0.008 1.06 1.01–1.11 0.013  CCR5 density on CD8+ T-cells 0.98 0.96–1.00 0.020 0.97 0.95–1.00 0.013 0.98 0.96–1.00 0.027 0.97 0.95–1.00 0.021  % ICOS+ CD8+ T-cells 9.87 2.06–47.25 0.004 7.26 1.46–36.06 0.015 9.85 2.02–48.13 0.005 7.74 1.51–39.58 0.014 Infant subsets  CCR5 density on CD4+ T-cells 1.04 1.01–1.06 0.003 1.03 1.01–1.06 0.009 1.04 1.01–1.07 0.002 1.04 1.01–1.06 0.008  CCR5 density on CD8+ T-cells 1.02 1.00–1.03 0.008 1.02 1.00–1.03 0.025 1.02 1.01–1.03 0.007 1.02 1.0–1.03 0.025  % CCR5+ CD4+ T-cells 2.11 1.17–3.83 0.013 1.92 1.05–3.50 0.031 2.11 1.16–3.84 0.014 1.92 1.06–3.5 0.031  % CCR5+ CD8+ T-cells Undefinedc  % HLA-DR+ CD8+ T-cells Undefinedc  % TIGIT+ CD4+ T-cells 1.86 1.06–3.26 0.031 2.25 1.12–4.50 0.022 1.83 1.04–3.23 0.037 2.36 1.17–4.78 0.017  % TIGIT+ CD8+ T-cells Undefinedc  % PD-1+ CD4+ T-cells 1.75 1.12–2.73 0.014 1.77 1.09–2.87 0.021 1.74 1.11–2.74 0.016 1.80 1.13–2.87 0.014  % PD-1+ CD8+ T-cells Undefinedc

Statistically significant results are shown in bold. CI, confidence interval.

aModel 1 examines maternal subsets (which were significantly different between mothers who did and did not transmit (Fig. 1), shown individually unadjusted (column 1), then adjusted for maternal viral load (column 2), maternal CD4+ T-cell counts (column 3) and both maternal viral load and CD4+ T cell counts simultaneously (column 4). These five subsets were combined in another model (i.e. takes into account all the subsets together), which after adjustment for both maternal viral load and CD4+ T-cell counts identified the three subsets that associated significantly with transmission and were used in model 2.

bModel 2 includes three maternal subsets shown together unadjusted (column 1), then adjusted for maternal viral load (column 2), maternal CD4+ T-cell counts (column 3) and both maternal viral load and CD4+ T-cell counts (column 4).

cCompletely distinguished between the groups.

Comparison of infants who did or did not acquire HIV-1

Compared with infants without HIV-1, infants with HIV-1 had a higher CCR5 density on CD4+ and CD8+ T-cells, higher frequencies of CCR5+, TIGIT+, PD-1+ CD4+ and CD8+ T-cells as well as higher frequencies of HLA-DR+ CD8+ T-cells (Fig. 1).

After adjusting for maternal viral load and maternal CD4+ T-cell count, CCR5 density on CD4+ T and CD8+ T-cells, and frequencies of CCR5+, TIGIT+, and PD-1+ CD4+ T-cells remained significantly associated with increased risk for acquisition of infection (Table 1). We could not undertake adjusted analyses for frequencies of CCR5+, HLA-DR+, TIGIT+, and PD-1+ CD8+ T-cells as these markers were so strongly related to infection, they almost completely distinguished between the groups.

Of the infant T-cell subsets that were significantly different between infants with and without HIV-1, CCR5 density on CD4+ T-cells, frequency of CCR5+ and TIGIT+ CD8+ T-cells correlated positively with infant viral load and frequency of CCR5+ and TIGIT+ CD8+ T-cells correlated negatively with infant CD4+ T-cell percentage (Supplemental Figure 3, https://links.lww.com/QAD/D121).

There were no sex-based differences in CCR5+ CD4+ or CD8+ T-cell density or in the frequency of CD4+ or CD8+ T-cell subsets expressing immune activation (CCR5, ICOS and HLA-DR) or immune checkpoint molecules (TIGIT and PD-1) (data not shown).

Influence of maternal antiretroviral treatment

There were no significant differences in maternal CCR5 density, maternal or infant viral load, or sex of the infants born to transmitting mothers who did or did not receive ART during pregnancy, or in infant CCR5 density, maternal and infant viral load according to duration of ART (Supplemental Figures 4 and 5, https://links.lww.com/QAD/D121).

Comparison of CCR5 density on HLA-DR-, HLA-DR+, and TIGIT− and TIGIT+ subsets

We repeated the comparisons between the groups re-analysing CCR5 density on HLA-DR- and HLA-DR+ and on TIGIT− and TIGIT+ CD4+ and CD8+ T-cells, separately. CCR5 density was higher on HLA-DR+ than HLA-DR- CD4+ and CD8+ T-cells in both mothers and infants, as has been shown previously [35]. Additionally, CCR5 density was higher on TIGIT+ than on TIGIT- CD4+ and CD8+ T-cells in the infants (Fig. 2).

Fig. 2:

CCR5 density on total CD4+ and CD8+ T-cells and HLA-DR−, HLA-DR+, TIGIT− and TIGIT+ CD4+ and CD8+ T-cells.

CCR5 density was higher in mothers who transmitted than in mothers who did not, and in infants with HIV-1 compared with infants without HIV-1 even on these subsets. Additionally, there was a larger spread of values for CCR5 density in mothers who transmitted and infants who acquired infection compared with mothers who did not transmit and infants without HIV-1 (Fig. 2).

After adjusting for maternal viral load and CD4+ T-cell count, maternal CCR5 density on HLA-DR-, HLA-DR+, TIGIT− and TIGIT+ CD4+ T-cells remained associated with transmission status. Similarly, infant CCR5 density on all infant subsets in the model, with the exception of CCR5 density on HLA-DR+ CD8+ T-cells, remained associated with acquisition of infection after adjusting for maternal viral load and CD4+ T-cell count (Supplemental Table 2, https://links.lww.com/QAD/D121).

Comparison of CCR5 density phenotypes in mother–infant pairs

Given that higher CCR5 density on both maternal and infant CD4+ T-cells was associated with HIV-1 transmission/acquisition, we postulated that mother–infant pairs sharing a combined high CCR5 density phenotype would have the highest transmission risk compared with the opposite extreme of a shared low CCR5 density phenotype. To test this hypothesis, we divided mother–infant pairs into four groups according to levels of CCR5 density above (high) or below (low) the medians calculated for all mothers and all infants – high mother high infant (Hh), low mother high infant (Lh), high mother low infant (Hl) and low mother low infant (Ll). Mother–infant pairs in which the infant was infected were significantly more likely to have the Hh phenotype (51.9 vs. 8%) and the Lh phenotype (29.6 vs. 12%) and less likely to have the Ll phenotype (7.4 vs. 52%) (P < 0.0001) (Fig. 3). The highest odds ratio associated with HIV-1 infection in the infant was associated with the Hh phenotype (odds ratio = 45.5, 95% CI 5.6–372), but the Lh phenotype was also over represented (OR = 17.3, 95% CI 2.4–127) compared with Ll phenotype as the reference. Similar results were observed after adjusting for maternal viral load and CD4+ T-cell counts (Table 2).

Fig. 3:

Classification of NTM/HEU and TM/HIV mother–infant pairs into four CCR5 density groups.

Table 2 - Logistic regression results predicting transmitter status comparing low mother low infant (Ll) to high mother high infant (Hh), low mother high infant (Lh) and high mother low infant (Hl) unadjusted and adjusted for maternal viral load and CD4+ T-cell counts. Unadjusted Adjusted for maternal VL Adjusted for maternal CD4+ T-cell counts Adjusted for maternal VL and CD4+ T-cell counts Subset Odds ratio 95% CI P Odds ratio 95% CI P Odds ratio 95% CI P Odds ratio 95% CI P Ll 1.0 – – 1.0 – – 1.0 – – 1.0 – – Hh 45.5 5.6–372 <0.001 28.8 3.2–263 0.003 60.3 6.3–580 <0.001 32.7 3.4–317 0.003 Lh 17.3 2.4–127 0.005 8.7 1–72 0.045 23.2 2.8–194 0.004 11 1.2–103 0.036 Hl 2.8 0.4–20.8 0.318 1.5 0.16–14 0.713 2.2 0.28–17 0.456 1.4 0.1–13 0.783

Statistically significant results are shown in bold. CI, confidence interval.

Discussion

Vertical transmission of HIV-1 is an ideal model for advancing our understanding of factors influencing both maternal transmissibility and infant susceptibility [36,37]. We hypothesized that CCR5 density on CD4+ T-cells would be an important measure of risk of HIV-1 intrauterine transmission/acquisition. Compared with mothers who did not transmit HIV-1, those who transmitted HIV-1 had higher CCR5 density on CD4+ and CD8+ T-cells and higher frequencies of CCR5, ICOS, and TIGIT expressing CD8+ T-cells. Similarly, compared with infants without HIV-1, infants with HIV-1 had higher CCR5 density on CD4+ and CD8+ T-cells, higher frequencies of CCR5, TIGIT, and PD-1 expressing CD4+ and CD8+ T-cells, and higher frequencies of HLA-DR+ CD8+ T-cells. Furthermore, a combined maternal–infant high CCR5 density phenotype increased the risk of transmission/acquisition of in-utero HIV-1 infection.

Increased CCR5 density correlates with high viral loads in PWH [35] and high maternal viral load is the strongest risk factor for maternal–infant HIV-1 transmission [38]. The high maternal ART coverage in this study may have led to a larger proportion of early in-utero infections occurring before the initiation of ART during pregnancy. CCR5 density on CD4+ T-cells remained associated with increased risk for in-utero HIV-1 transmission/acquisition even after adjusting for maternal viral load and CD4+ T-cell count. The increased frequencies of ICOS+CD8+ T-cells in the HIV-1 transmitting mothers suggests an association between maternal immune activation and increased risk of HIV-1 transmission as previously shown using CD38 as the activation marker [39]. In our study, ICOS was a stronger marker of activation in the mothers who transmitted than was HLA-DR.

As expected, infants with HIV-1 had higher levels of immune activation (HLA-DR+ CD8+ T-cells) confirming previous studies [39–41]. This was not evident with ICOS as an activation marker. Our finding of higher levels of CCR5 in infants with HIV-1 corroborates studies showing that CCR5 density influences in-vitro infectibility [42] and that individuals with high CCR5 density on CD4+ T-cells are more susceptible to HIV-1 infection [15].

When comparing the mothers to their infants, we observed that, as expected [43], CCR5 expression (density and frequency) was lower in infants compared with their mothers, regardless of infection status. CCR5 expression is constitutive, but immune activation related to environmental factors can increase this expression