Binding and Neutralizing Capacity of Respiratory Syncytial Virus (RSV)-Specific Recombinant IgG Against RSV in Human Milk, Gastric and Intestinal Fluids from Infants

Abstract

Oral administration of pathogen-specific recombinant antibodies may help to prevent infant gastrointestinal (GI) pathogen infection; however, to neutralize an infectious agent, these antibodies must resist degradation in the GI tract. Palivizumab, a recombinant antibody specific for the respiratory syncytial virus (RSV), was used as a model for pathogen-specific IgG in human milk. The aim was to compare the remaining binding capacity of palivizumab in milk between three mothers after exposure to an in vitro model of infant gastrointestinal digestion (gastric and duodenal fluids) using ELISA. The neutralizing capacity of palivizumab in pooled human milk, gastric contents, and stools from preterm infants was also evaluated for blocking RSV with green fluorescent protein (RSV-GFP) infection in Hep-2 cells using confocal and inverted microscopy and flow cytometry. The reduction of palivizumab binding capacity in human milk and digested samples was slightly different between mothers. Overall, palivizumab decreased 50% after simulated gastric digestion with pepsin and 62% after simulated intestinal digestion with pancreatin. Palivizumab (2-8 μg/mL) in human milk or stool samples blocked RSV (3.4 × 10⁴ FFU/mL) infection (no syncytia formation on Hep-2 cells) by microscopy. Syncytia formation was detected on Hep-2 cells when RSV was incubated in gastric contents or virus medium with 2-4 μg/mL of palivizumab, but no infection was observed at 8 μg/mL. No fluorescence (absence of infected cells) was detected when palivizumab (100 μg/mL) was incubated in human milk or medium with RSV-GFP (1.1 × 10⁵ FFU/mL), whereas fluorescence increased with the reduced concentration of palivizumab using flow cytometry. These results suggest that undigested and digested matrices could change the binding and neutralizing capacity of viral pathogen-specific antibodies.

Keywords: neonatal infection; passive immunity; pathogen-specific antibody.

Figure 1

Illustration of palivizumab ELISA. (A) Anti-idiotypic Fab antibody is coated in 96-well plate and can specifically recognize (B) the monoclonal antibody palivizumab IgG1 in biological samples. (C) After washing the biological samples, human anti-palivizumab IgG1 conjugated with horseradish peroxidase (HRP) is added to detect palivizumab.

Figure 2

Binding capacity of palivizumab in human milk with different conditions. (A) Palivizumab in human milk; non-treated (NT), incubated at 37 °C for 1 h (INC37), heat-treated at 55 °C for 30 min (HT55) and at 100 °C for 30 min (HT100), and frozen and thawed at –20 °C (F/T20) and at –80 °C (F/T80). Values are mean ± SD, n = 3. (B) Palivizumab in pooled mother’s milk samples incubated at 37 °C for 2 h (INC37) and measured every 30 min. Values are mean ± SD, n = 6. Asterisks show statistically significant differences between groups (*** p < 0.001; ** p < 0.01; * p < 0.05) using one-way ANOVA followed by Dunnett’s multiple comparison tests.

Figure 3

Palivizumab stability across simulated term infant digestion from three different mother’s milk samples. Palivizumab in the average of three mother’s milk (HM) samples; in gastric conditions at 0 min (G0 – P), at 30 min (G30 - P) and at 60 min (G60 – P) without protease (pepsin); in intestinal conditions at 0 min (G60 + I0 – P), at 30 min (G60 + I30 – P), 60 min (G60 + I60 – P), 90 min (G60 + I90 – P) and 120 min (G60 + I120 – P) without protease (pancreatin). Values are mean ± SD from 3 mother’s milk samples with 6 replicates for each condition. Letters a, b, c and d show statistically significant differences between groups (p < 0.05) using one-way ANOVA followed by Tukey’s multiple comparison tests.

Figure 4

Effect of bile salts and pH on palivizumab stability. (A) Effect of bile salts on palivizumab stability at 0 h and after incubation (2 h) at 37 °C with shaking at 300 rpm in presence or in absence of bile salts and/or milk. HM, milk with no treatment; HM + bile, milk and bile; Bile + PBS, bile in PBS; and PBS, PBS alone. Values are mean ± SD from pooled mother’s milk samples with 3 replicates at each of 2 dilutions. Two-way ANOVA followed by Tukey’s multiple comparisons test where means were compared between time 0 and 2 h for each sample. (B) Effect of pH on palivizumab stability at 0 h and 2 h at 37 °C with shaking at 300 rpm with pH adjusted to 4, 7, and 8. HM pH 4, milk adjusted pH to 4; HM pH 7, milk adjusted pH to 7; and HM pH 8, milk adjusted pH to 8. Values are mean ± SD from 3 mother’s milk samples with 3 replicates at each of 2 dilutions. Two-way ANOVA followed by Tukey’s multiple comparisons test where means were compared between time 0 and 2 h for each sample.

Figure 5

Neutralizing palivizumab IgG against the human respiratory syncytial virus with a green fluorescent protein (RSV-GFP) in pooled human milk using confocal microscopy. RSV-GFP suspension containing 50% tissue culture infectious dose (TCID50) (3.4 × 10⁴ focus forming units (FFU)/mL) was added to pooled human milk (filtered supernatant diluted in 1:10) with 0 and 8 μg/mL of palivizumab for 2 h at 37 °C. The mixtures were transferred to confluent Hep-2 cells for 2 h at 37 °C. A blue fluorescent nucleic acid stain, 4,6-diamidino-2-phynylindole dilactate (DAPI) was added to perform an immunofluorescence staining of Hep-2. GFP expression in infected cells was monitored using a confocal microscope system (Zeiss LSM 780 NLO, White Plains, NY, USA) at 64× objective. Scale bar = 100 μm).

Figure 6

Neutralizing capacity of palivizumab IgG against the human respiratory syncytial virus with a green fluorescent protein (RSV-GFP) in pooled human milk, pooled gastric contents and pooled stool from preterm infants using inverted microscopy. RSV-GFP suspension containing 50% tissue culture infectious dose (TCID50: 3.41 × 10⁴ focus forming units (FFU)/mL) was added in virus medium and filtered supernatant of diluted pooled human milk (1:10), pooled gastric contents (1:20) or pooled stool samples (0.2 g/mL and 1:25) with 0, 2, 4, and 8 μg/mL of palivizumab for 2 h at 37 °C, 5% CO₂. The mixtures were transferred to confluent Hep-2 and incubated for 2 h at 37 °C. The neutralizing capacity of palivizumab in each condition was monitoring using an Olympus IX51 fitted with a Q Image camera (20× objective) to compare the cytopathic effects. Palivizumab at (A) 8 μg/mL, (B) 4 μg/mL, (C) 2 μg/mL, (D) 0 μg/mL in virus medium with RSV and (E) at 8 ug/mL without RSV in virus medium. Palivizumab at (F) 8 μg/mL, (G) 4 μg/mL, (H) 2 μg/mL, (I) 0 μg/mL in pooled human milk with RSV, and (J) at 8 ug/mL without RSV in pooled human milk. Palivizumab at (K) 8 μg/mL, (L) 4 μg/mL, (M) 2 μg/mL, (O) 0 μg/mL in pooled gastric contents from preterm infants with RSV and (P) at 8 ug/mL without RSV. Palivizumab at (Q) 8 μg/mL, (R) 4 μg/mL, (S) 2 μg/mL, (T) 0 μg/mL in stools from preterm infants with RSV and (U) at 8 ug/mL without RSV.

Figure 7

Neutralizing capacity of palivizumab IgG against the human respiratory syncytial virus with a green fluorescent protein (RSV-GFP) in pooled human milk and virus medium using flow cytometry. RSV-GFP suspension (1.1 × 10⁵ focus forming units (FFA)/mL) was added in virus medium and filtered supernatant of diluted pooled human milk (1:10) with 0, 25, 50, and 100 μg/mL of palivizumab for 2 h at 37 °C, 5% CO₂. The mixtures were transferred to confluent Hep-2 and incubated for 2 h at 37 °C. Fluorescence measured by flow cytometry (FACSCalibur) in Hep-2 cells incubated with RSV-GFP (1.1 × 105 FFA/mL) (A) in virus medium with 0 and 100 μg/mL of palivizumab after 3 days of infection; and (B) at day 1, day 2, and day 3 of infection in the absence of palivizumab (neg Ctl: no RSV and no palivizumab). (C) Fluorescence measured by flow cytometry in the presence of RSV-GFP in human milk or virus medium with palivizumab (0, 25, 50 and 100 μg/mL). *** p < 0.001; ** p < 0.01; * p < 0.05. (D) Percentage of GFP-expressed Hep-2 cells (or infected cells) in the presence of RSV-GFP in human milk or virus medium with palivizumab.