Human Cytomegalovirus Reactivation During Lactation: Impact of Antibody Kinetics and Neutralization in Blood and Breast Milk

Abstract

Human cytomegalovirus (HCMV) is shed into breast milk in nearly every seropositive woman during lactation. This reactivation shows mostly a self-limited, unimodal course. The dynamics and functional role of HCMV-specific-IgG in breast milk and in plasma during reactivation are unknown. Milk whey viral loads were monitored with real-time PCR in 18 HCMV-seropositive mothers over two months postpartum. HCMV-antibody binding assays (ECLIA) and antigen-specific immunoblotting were performed from plasma and corresponding milk samples. Epithelial-cell-specific neutralization was used to analyze functional antibodies in plasma- and whey-pools. Viral loads in milk whey showed unimodal courses in 15 of 18 mothers with peak viral loads around one month postpartum. HCMV-specific-IgG-antibodies increased significantly in plasma and milk whey during reactivation. The mean levels of plasma IgG were about 275-fold higher than in whey. Only antibodies against tegument protein p150 were continuously expressed in both compartments. Anti-glycoprotein-B1 IgG-antibodies were variably expressed in whey, but continuously in plasma. Neutralization assays showed 40-fold higher NT-50 values in plasma compared to whey at two months postpartum. During reactivation, HCMV-specific-IgG reactivities and neutralizing capacities are much lower in whey than in plasma. Therefore, their specific role in the decrease and discontinuation of virus-shedding in milk remains unclear.

Keywords: HCMV; IgG; breastfeeding; glycoprotein B; milk whey; neutralization; p150; recomLine blot.

Figure 1

Milk whey viral loads (target region UL83) of 17 of 18 HCMV-seropositive BlooMil study mothers at T1 (10 to 15 days pp), T2 (25 to 30 days pp), T3 (40 to 45 days pp) and T4 (55 to 60 days pp). The insert shows low viral loads (peak levels ≤ 6x10⁴ copies/mL). One mother did not reactivate during observation. Individual color identification.

Figure 2

HCMV-specific-IgG ECLIA values (mean + 95%CI) of 18 seropositive mothers of the BlooMil study in (A) plasma and (B) milk whey from T1 (10 to 15 days pp), T2 (25 to 30 days pp), T3 (40 to 45 days pp) and T4 (55 to 60 days pp). The Friedman test was used for the kinetics and showed a significant change in plasma from T1 to T4 p < 0.001 and T1 to T3 p = 0.003, but in whey, no significant change was detected. °The Wilcoxon matched-pairs signed rank test was used in whey to compare single time ranges; no correction was used. Individual color identification as in Figure 1. Correlation of plasma and milk whey HCMV-specific-IgG were determined by Spearman’s rank correlation and non-linear regression at (C) T1 with r = 0.75, p = 0.004 and (D) T4 with r = 0.57, p = 0.045.

Figure 3

Reactivity scores of IgG recomLine blots for anti-recIE1-IgG at (A) T1 (10 to 15 days pp) and (B) T3/T4 (40 to 60 days pp) and anti-recCM2-IgG at (C) T1 (10 to 15 days pp) and (D) T3/T4 (40 to 60 days pp). The X-axis shows the number of study participants. Nr: non-reactive, +/-: lower as cut-off, but detectable, +: one-fold, ++: two-fold, +++: three-fold stronger than cut-off reactivity. Breast milk was diluted two-fold, plasma 100-fold.

Figure 4

Reactivity scores of IgG recomLine blots for anti-rec150-IgG at (A) T1 (10 to 15 days pp) and (B) T3/T4 (40 to 60 days pp) and anti-recp65-IgG at (C) T1 (10 to 15 days pp) and (D) T3/T4 (40 to 60 days pp). Scoring of antibody reactivity as in Figure 3.

Figure 5

Reactivity scores of IgG recomLine blots for anti-recgB1-IgG at (A) T1 (10 to 15 days pp) and (B) T3/T4 (40 to 60 days pp) and anti-recgB2 at (C) T1 (10 to 15 days pp) and (D) T3/T4 (40 to 60 days pp). Scoring of antibody reactivity as in Figure 3.

Figure 6

Neutralization assays of HCMV-seropositive and -negative pools using ARPE-19 target cells. (A) Plasma and (B) whey immediate early antigen (IEA) plaque counts in a two-fold serial dilution (mean+SD). HCMV-IgG calibration using hyperimmunoglobulin (HIG) preparation (prediluted 1:700 to mean ECLIA values of whey and 1:6.5 for plasma values). Neutralization (NT)-capacity in (C) plasma and in (D) whey were calculated by using the HCMV-seronegative pools as 100% reference for every dilution step. NT-50 values were calculated with a Probit analysis of the seropositive pool with mean values of the seronegative pools as reference.