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Background Vertical transmission of Group B Streptococcus (GBS) is a prerequisite for early-onset disease and a consequence of maternal GBS colonization. Disease protection is associated with maternally-derived anti-GBS antibody. Using a novel antibody-mediated C3b/iC3b deposition flow cytometry assay which correlates with opsonic killing we developed a model to assess the impact of maternally-derived functional anti-GBS antibody on infant GBS colonization from birth to day 60–89 of life. Methods Rectovaginal swabs and cord blood (birth) and infant nasopharyngeal/rectal swabs (birth, day 6 and day 60–89) were obtained from 750 mother/infant pairs. Antibody-mediated C3b/iC3b deposition with cord and infant sera was measured by flow cytometry. Results We established that as maternally-derived anti-GBS functional antibody increases, infant colonization decreases at birth and up to three months of life, the critical time window for the development of GBS disease. Further, we observed a serotype (ST)-dependent threshold above which no infant was colonized at birth. Functional antibody above the upper 95th confidence interval for the geometric mean concentration was associated with absence of infant GBS colonization at birth for STII (p < 0.001), STIII (p = 0.01) and STV (p < 0.001). Increased functional antibody was also associated with clearance of GBS between birth and day 60–89. Conclusions Higher concentrations of maternally-derived antibody-mediated complement deposition are associated with a decreased risk of GBS colonization in infants up to day 60–89 of life. Our findings are of relevance to establish thresholds for protection following vaccination of pregnant women with future GBS vaccines.
We undertook a prospective longitudinal cohort study in two government health centers offering antenatal care to women in the Fajara area of coastal Gambia, a low-income country with an annual birth rate of 43.1/1000 population, neonatal sepsis rate of 4.4/1000 live births [7] and neonatal mortality rate of 28/1000 live births [8]. The study was approved by the joint Gambian Government/Medical Research Council Research Ethics Committee, SCC 1350 V4. Eligibility and recruitment details have been previously described [9]. Participants were followed up daily at home for 6 days and then asked to return to clinic when the infant was 60–89 days old for final follow up and vaccinations. Rectovaginal swabs were taken from enrolled women presenting in labor and cord blood was taken after delivery but prior to separation of the placenta. Nasopharyngeal and rectal swabs were taken from all eligible infants at four hours. Nasopharyngeal and rectal swabs were also taken from infants at day 6 of life and again at 60 to 89 days of life together with an infant serum sample. An infant was deemed to be colonized if either rectal or nasopharyngeal swabs were positive for GBS (or both). Colonization at or after day 6 was defined as rectal colonization as GBS is unlikely to remain a true colonizer of the nasopharynx. Persistent colonization was defined as swab-culture positive for GBS at all three time points; intermittent colonization was defined as swab-culture positive for GBS on two occasions at either birth and day 6 or birth and day 60–89 or day 6 and day 60–89, one time point is defined as swab culture positive for GBS at either birth or day 6 or day 60–89. The sites and number of infants colonized have been reported elsewhere [9]. All swabs were collected in skim-milk tryptone glucose glycerol (STGG) transport medium, stored at 4 °C and transported to the Medical Research Council laboratories, The Gambia within 4 hours of collection. On arrival the samples were vortexed briefly and immediately frozen at −70 °C until processing. All swab specimens were then inoculated into Todd-Hewitt broth supplemented with colistin and naladixic acid and were processed for isolation of GBS using standard laboratory procedures [10]. Presumptive positive GBS samples were identified by latex agglutination (Oxoid). All swabs were subjected to real-time polymerase chain reaction (PCR) [11]. All GBS positive isolates were then serotyped using conventional PCR and gel agarose electrophoresis [12]. Antibody-mediated C3b/iC3b deposition onto the surface of formaldehyde-fixed GBS was measured using a flow cytometric assay performed in 96-well microtitre plates [14]. Briefly, 35 μL serotype Ia, II, III or V GBS bacteria at 5 · 14 × 107 CFU/mL in blocking buffer (1% BSA in PBS) were added to 10 μL IgG-depleted human plasma as the complement source [15] and 5 μL of each test serum. Plates were incubated for 7.5 min at 37 °C with shaking (900 rpm), and the bacteria pelleted by centrifugation at 3000g for 5 min. Supernatant was removed and the bacteria washed once with 200 μL blocking buffer. Bacteria were resuspended in 200 μL blocking buffer containing 1:500 sheep anti-human C3c FITC (Abcam) and incubated for 20 min before washing and analysis by flow cytometry. The CDA values were compared to OPkA titres obtained for serotypes Ia, III and V using the HL-60 cell line as described [16], using strains 515 02/2012 (Ia), COH1 11/2013 (III) and CJBIII 03/2009 (V) (provided by Prof. Carol Baker, Baylor College of Medicine, Houston, USA). Briefly, heat-inactivated serum sample (12.5 μL) was mixed with 25 µl of bacteria revived from frozen stock (thawed and initially resuspended at 5 × 105 CFU/mL in HBSS and then diluted 1/2 in HBSS containing 10% baby rabbit complement to give 6250 CFU per well), of GBS strains, 75 μL HL60 cells at a concentration of 2.6 × 107 cells/mL and 12.5 µl IgG-depleted human complement [15]. Samples were incubated at 37 °C for 1 hour with shaking at 600 rpm in a Thermomixer (Eppendorf, Germany). Two positive controls and six negative controls were added to each plate. The following negative controls were used: two wells with bacteria, complement and phagocytes but without human serum; two wells with bacteria, positive control serum and complement but without phagocytes and two wells with bacteria, serum, phagocytes and heat inactivated complement. Before and after the one-hour incubation (T0 and T60), each reaction was diluted in sterile water to 1:20, 1:100 and 1:200. 10 μL of each dilution was subsequently plated by the tilt method onto blood agar plates and incubated overnight at 37 °C in 5% CO2. The opsonophagocytic activity was determined as the mean log10 reduction in GBS CFU/mL after 60 min of incubation at 37 °C compared to T0 (LogT0-LogT60). The lowest serum dilution analyzed was 1:30. Thus for statistical analysis, samples below the limit of detection were assigned an arbitrary titer of 15. For both the CDA and the OPkA, results were calibrated with standard serotype-specific monovalent vaccinee serum kindly provided by Prof. Carol Baker (as above). CDA results were expressed at geometric mean (GM) of the fluorescence intensity minus the complement-only control (FI-C′). The sample size was calculated on the basis of the previously observed 24% colonization rate [17], to provide at least 180 colonized women (95% confidence interval (CI) 150–202 women) and 90 colonized infants (95% CI 72–107 infants). The sample size of 180 colonized women was chosen to ensure at least 10 samples of the least prevalent serotype based on historical data from The Gambia (serotype III, 6%) [17], in order to allow longitudinal colonization analyses. Statistical analyses were completed using STATA version 14 (StataCorp 2014, Texas) and GraphPad Prism version 6·0 (GraphPad Software Inc, La Jolla, California). The operating characteristics of the CDA were assessed as a ‘test’ for correctly classifying the paired OPkA titer as <30 (<50% killing) or ≥30 (50% or greater killing observed). For each pairwise set of comparisons, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and positive and negative likelihood ratios (LR+ = sensitivity/1-specificity and LR− = 1-sensitivity/specificity) of the CDA were calculated. Potential differences in antibody-mediated C3b/iC3b deposition between sera from colonized and non-colonized mothers and infants were evaluated by one-way analysis of variance (ANOVA) after log transformation of data. Four groups were compared (mother colonized/infant non-colonized; mother colonized/infant colonized; mother non-colonized/infant colonized and neither mother nor infant colonized). The correlation between bacterial concentration and anti-GBS serotype-specific antibody was evaluated using Deming linear regression and 95% confidence intervals (GraphPad Software Inc, La Jolla, California). For all comparisons, p < 0.05 was considered to be significant. The funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to all data and the corresponding authors had final responsibility for the decision to submit for publication.
