MFGM-enriched whey displays antiviral activity against common pediatric viruses in vitro

Abstract

Background: Among the most common mucosal viral infections in infants are rotavirus, one of the main causes of severe gastroenteritis in infants and children up to 5 years, and respiratory syncytial virus (RSV), one of the leading causes of lower respiratory tract infections. Both human milk and bovine milk derived factors may provide protection against mucosal viral infections. More recently, a similar activity of milk derived proteins was suggested for SARS-CoV-2. The goal of the current study was to test antiviral activity of the bovine milkfat globule membrane (MFGM) against rotavirus, RSV and SARS-CoV-2 and to further characterize MFGM-enriched whey to identify which components in MFGM-enriched whey may contribute to the inhibitory activity.

Methods: The effects of MFGM-enriched whey, its whey protein isolate counterpart (WPI, obtained from the same production process) and a conventional whey protein concentrate (WPC) on rotavirus (strains Wa and SA114F), RSV (strain RSV-A2) and SARS-CoV-2 (Alpha variant) infectivity were determined using MA104 cells, human alveolar basal epithelial (A549) cells and monkey kidney (Vero E6) cells, respectively. The compounds were characterized in detail by LC-MS/MS and ³¹P-NMR to determine protein and phospholipid composition, respectively.

Results: Relative to its WPI counterpart, MFGM-enriched whey demonstrated a dose-dependent inhibition for both rotavirus and RSV whereas for SARS-CoV-2 inhibition was only observed at the highest concentration tested. Label-free quantification (LFQ) and intensity based absolute quantification (iBAQ) of identified proteins revealed a clear difference between MFGM-enriched whey and its controls including enrichment of known MFGM proteins and non-MFGM proteins that are enriched simultaneously, some of which have previously been demonstrated to display anti-viral activity. Although not completely absent from other whey protein preparations, MFGM-enriched whey had the highest specific and total phospholipid levels.

Conclusion: MFGM-enriched whey displayed antiviral activity against multiple viruses of clinical importance. This study provides insights into the active components in MFGM-enriched whey and may contribute to previous clinical observations with MFGM-enriched formula demonstrating reduced respiratory and gastrointestinal infections in formula fed infants.

Keywords: MFGM; RSV; SARS-CoV-2; milk; rotavirus.

Figure 1

MFGM-enriched whey dose-dependently inhibited rotavirus infection in MA104 cells. Inhibition of rotavirus infection, strain WA G1P[8], in MA104 cells by different concentrations of MFGM-enriched whey (A), MFGM-enriched whey (dark grey bars), the corresponding WPI (open bars) and WPC reference (light grey bars) (B). Inhibition of SA11-4F G3P[1] (open bars) and WA G1P[8] (dark grey bars) strains in MA104 cells by different concentration of MFGM-enriched whey (C). Means ± SEM significantly (p < 0.01) different from the media control (i.e., UT) are indicated with asterisks (**p < 0.01, ***p < 0.001). All measurements were performed twice in triplicate.

Figure 2

MFGM-enriched whey dose-dependently inhibited RSV infection in A549 cells. Inhibition of RSV-A2 in A549 cells by MFGM-enriched whey (dark grey bars) and the corresponding WPI (open bars) at different concentrations. Means ± SEM significantly (p < 0.01) different from the media control (i.e., UT) are indicated with asterisks (**p < 0.01, ***p < 0.001). All measurements were performed in triplicate.

Figure 3

MFGM-enriched whey reduced SARS-CoV-2 infection. Inhibition of SARS-CoV-2 in Vero E6 cells by MFGM-enriched whey (dark grey bars) and the corresponding WPI (open bars) at different concentrations. Means ± SEM significantly (p < 0.01) different from the media control (i.e., UT) are indicated with asterisks (**p < 0.01, ***p < 0.001). All measurements were performed in triplicate.

Figure 4

LC–MS/MS analyses LFQ (A) and iBAQ (C) intensity of top 20 most abundant proteins in MFGM-enriched whey (black bars, n = 3), the corresponding WPI (open bars, n = 3) and WPC reference (grey bars, n = 3). For a better visualization of less dominant proteins, LFQ (B) and iBAQ (D) intensity were also compared without including the two major whey proteins, alpha-lactalbumin and beta-lactoglobulin, in the dataset. Beta-lactoglobulin (P02754), Glycam-1 (P80195), Bovine serum albumin (P02769), Ig-like domain-containing protein (F1N160, F1MLW7), Ig-like domain-containing protein (A0A3Q1M3L6), Butyrophilin (P18892), Alpha-S1-casein (P02662), Lactadherin (Q95114), CD36 (P26201), Lactoferrin (P24627), Alpha-lactalbumin (P00711), Xanthine dehydrogenase (P80457), Fatty acid-binding protein (P10790), Kappa-casein (P02668), Ig-like domain-containing protein (G3N0V0, A0A3Q1N3I9), Polymeric immunoglobulin receptor (P81265), Ig-like domain-containing protein (G3MXB5, A0A3Q1LRW4), Perilipin-2 (Q9TUM6), Endopin 2 (A0A0A0MP92, A2I7N3), Lactoperoxidase (P80025).