The effect of whey protein on viral infection and replication of SARS-CoV-2 and pangolin coronavirus in vitro

Abstract

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been detected in human breastmilk, infants' safety with breastmilk feeding is of great concern for women with coronavirus disease 2019 (COVID-19). 1 It is known that milk has antiviral properties. 2 However, little is known about the antiviral property of human breastmilk to SARS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that whey protein from human breastmilk effectively inhibited both SARS-CoV-2 and GX_P2V by blocking viral attachment and viral replication at entry and even post entry. Moreover, human whey protein inhibited infectious virus production, as proved by the plaque assay. We found that whey protein from different species, such as cow and goat, also showed anti-coronavirus properties. Commercial bovine formula milk also showed similar anti-SARS-CoV-2 activity. Firstly, healthy human breastmilk samples collected in 2017 and stored properly at −80°C were tested for their potential effects on SARS-CoV-2 infection. Mothers provided informed consent. This study was approved by the ethics committees of the Medical Center and all samples were anonymized. The skimmed breastmilk was obtained after removal of the lipid fraction. Vero E6 cells were infected with a mixture of SARS-CoV-2 pseudovirus (650 TCID 50 /well) and human breastmilk (4 mg/ ml). Human breastmilk from eight donors showed a significant inhibition of more than 98% of the SARS-CoV-2 pseudovirus. As reported recently, a SARS-CoV-2-related pangolin coronavirus model (GX_P2V) 3 shares 92.2% amino acid identity in spike protein with SARS-CoV-2, which is a suitable model for SARS-CoV-2 infection research. We utilized GX_P2V (MOI: 0.01 in Vero E6 cells) as the model to study the effect of breastmilk on viral infection and also found similar results (Fig. 1a). The inhibition is concentration dependent with an EC 50 of 0.13 mg/ml of total protein (Fig. 1b and Supplementary Fig. S1) in the SARS-CoV-2 pseudovirus model. Consistent with the SARS-CoV-2 study, the GX_P2V model also showed inhibition with an EC 50 of about 0.5 mg/ml of total protein (Fig. 1c and Supplementary Fig. S2). Interestingly, human breastmilk did not show any cytotoxicity to Vero E6 cells (CC 50 > 3 mg/ml), and even promoted cell proliferation. These results indicated that human breastmilk showed high anti-SARS-CoV-2 and anti-GX_P2V property, but limited cytotoxicity to Vero E6 cells. We then assessed the impact of human breastmilk on infectious virus production in Vero E6. RT-qPCR analysis of the GX_P2V virus from supernatant showed that even 0.16 mg/ml of breastmilk significantly blocked viral production (Fig. 1d). Western blot of viral nucleoprotein also showed similar results (Fig. 1e). To investigate the infectious virus, we performed plaque assay. As shown in Fig. 1f, the plaque assays showed that live viruses were significantly lower in breastmilk treatment compared to the control group, which confirmed that

Consequences of Early Separation of Maternal-Newborn Dyad in Neonates Born to SARS-CoV-2 Positive Mothers: An Observational Study

“…Recent evidence indicates that breastfeeding does not seem to be associated with neonatal SARS-CoV-2 infection because viral transmission through the milk, if any, should be rare [23] and because a robust sIgA-dominant SARS-CoV-2 antibody response is detectable in human milk soon after infection in a significant majority of individuals [24], suggesting a possible protection granted by the mothers to their infants [25][26][27][28][29][30]. Moreover, there is evidence in vitro suggesting whey proteins in human breastmilk as a direct-acting inhibitor of SARS-CoV-2 infection and replication [31]. While it was demonstrated that a healthy mother-infant dyadic process positively impacts the infants' behavioral, cognitive, and socioemotional development [32], the possible long term effects of neonatal SARS-CoV-2 early infection, including neurological implications, are obviously unknown [33].…”

Mobile-Assisted Virtual Bonding Enables Breast Milk Supply in Critically Ill Mothers With COVID-19: A Reflection on the Feasibility of Telelactation

“…It is worth commenting that all the nurses who took part in this initiative provided excellent feedback (even though some of them had initial apprehensions), and now the Mazandaran regional maternity unit offers a telelactation program to mothers who remain in isolation with infants admitted to NICU. Recent reports about the inhibitory capacity of breast milk against COVID-19 [ 18 ] give additional impetus to our approach, which has the potential transferability to resource-limited settings. The previous experience of the unit in achieving induction of lactation in challenging clinical scenarios through international collaborative efforts [ 19 ] also provided confidence to our neonatal team to pursue a low-cost mobile phone-enabled e-lactation in the midst of a pandemic.…”

Anti-Infective, Anti-Inflammatory, and Immunomodulatory Properties of Breast Milk Factors for the Protection of Infants in the Pandemic From COVID-19

“…However, other researchers have verified that the main antimicrobial components of breast milk, such as LF and IgA antibody, showed limited anti-coronavirus activity, but whey protein is a direct-acting inhibitor of SARS-CoV-2 infection by blocking viral attachment, entry, and even post-entry viral replication. This suggests that other factors of breast milk may play the important anti-COVID-19 role (243). Even with the limits of a preliminary and preprint report, and with the necessity of further investigation of its molecular mechanism of action in the context of SARS-CoV-2 infection, the data are interesting.…”