key: cord-0328624-1zd3hjpo authors: Lai, Xinyuan; Yu, Yanying; Xian, Wei; Ye, Fei; Ju, Xiaohui; Luo, Yuqian; Dong, Huijun; Zhou, Yihua; Tan, Wenjie; Zhuang, Hui; Li, Tong; Liu, Xiaoyun; Ding, Qiang; Xiang, Kuanhui title: Inhibition of SAR S-CoV-2 infection and replication by lactoferrin, MUC1 and α-lactalbumin identified in human breastmilk date: 2021-10-29 journal: bioRxiv DOI: 10.1101/2021.10.29.466402 sha: a46ff12f582c9c74624bf272f495d710f93ab978 doc_id: 328624 cord_uid: 1zd3hjpo The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to the public health. Human breastmilk is an extremely complex with nutritional composition to nourish infants and protect them from different kinds of infection diseases and also SARS-CoV-2 infection. Previous studies have found that breastmilk exhibited potent antiviral activity against SARS-CoV-2 infection. However, it is still unknown which component(s) in the breastmilk is responsible for its antiviral activity. Here, we identified Lactoferrin (LF), MUC1 and α-Lactalbumin (α-LA) from human breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and in vitro confirmation that inhibited SARS-CoV-2 infection and analyzed their antiviral activity using the SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) in the Huh7.5, Vero E6 and Caco-2-N cell lines. Additionally, we found that LF and MUC1 could inhibit viral attachment, entry and post-entry replication, while α-LA just inhibit viral attachment and entry. Importantly, LF, MUC1 and α-LA possess potent antiviral activities towards not only wild-type but also variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.1 (kappa). Moreover, LF from other species (e.g., bovine and goat) is still capable of blocking viral attachment to cellular heparan sulfate. Taken together, our study provided the first line of evidence that human breastmilk components (LF, MUC1 and α-LA) are promising therapeutic candidates warranting further development or treatingVID-19 given their exceedingly safety levels. It confers a great threat to global public health since the pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since outbreak in the late of 2019, which showed close phylogenetic relationship with SARS-CoV outbreak in 2002 1 . The high rates of human deaths and pandemic distribution worldwide have caused the collapse of health systems in many countries, especially in developing countries 2 . In addition, the ongoing pandemic has resulted in several viral variants [3] [4] [5] . These variants may potentially alter viral transmission, pathogenicity, efficacy of drug treatment as well as the binding capacity of vaccine elicited antibodies [6] [7] [8] [9] . In this situation, it raises the need of several effective drugs with low toxicity to treat this disease. As reported previously, COVID-19 pandemic also confers great concern of motherto-child transmission (MTCT) by breastfeeding [10] [11] [12] [13] [14] . In addition to the report of SARS-CoV-2 RNA was detected in human breastmilk, it was still unclear that SARS-CoV-2 could transmit to infants through breastfeeding 13 . Several evidences in the clinic study showed that SARS-CoV-2 couldn't transmit to infants by breastfeeding [10] [11] [12] 15, 16 . However, it is controversial if live SARS-CoV-2 existing in the breastmilk could still be infectious. Human milk is uniquely suited to breastfeed human infants due to its nutritional composition and bioactive factors for promoting antimicrobial and immunomodulatory effects 17 . Breastmilk could inhibit several viruses infection, such as human immunodeficiency virus (HIV), cytomegalovirus (CMV) and dengue virus. It was reported that breastmilk could not only inhibit several enveloped viruses such as herpes simplex types 1 and 2, HIV and CMV, but also showed effective activity in vitro against many non-enveloped viruses like rotavirus, enterovirus and adenovirus 17, 18 . The lactoferrin (LF), a important component in breastmilk, could suppress SARS-CoV and SARS-CoV-2 through blocking virus to bind heparan sulfate proteoglycans, raising a concern that human breastmilk can also suppress SARS-CoV-2 infection [19] [20] [21] . Our previous study had confirmed that human breastmilk significantly inhibits SARS-CoV-2 and its related pangolin coronavirus (GX_P2V) infection and have strong potent anti-SARS-CoV-2 effects than LF treatment alone, indicating that there are potential other factors in breastmilk also play important role in inhibiting SARS-CoV-2 inhibition and replication 22 . Thus, it is still unclear that which components are potential to suppress SARS-CoV-2 infection in an overall study. Breastmilk is an extremely complex to nourish infants and protect them from different kinds of disease. It contains more than 400 different proteins and many of them exhibit the antimicrobial activity 23 . Proteins in human milk can be divided into three groups, caseins, mucin and whey proteins. These bioactive proteins from the whey fraction include LF, mucins (MUC1and MUC4), α-lactalbumin (α-LA), lactadherin, lactoperoxidase, IgA and lysozyme (LZ) and so on 23, 24 . LF is an iron-binding protein with two molecules of Fe 3+ per protein and rich in human milk (1-2 g/L in mature milk, 5-10 g/L in colostrum), which relatively lower in other species such as bovine milk (0.02-0.3g/L in mature milk, 2-5 g/L in colostrum) 24 . As reported previously, LF is an important component to protect infants from microbes and virus infection 17, 24 . LZ is also known to be a key protein inhibiting bacteria and rich in human milk (0.4g/L). Lactadherin, a 46 KDa mucin associated protein, was reported that it displayed high viral receptor binding and showed specific anti-rotavirus activity 24 . MUC1 has the antiviral activity to inhibit influenza virus infection 25 . In addition, human milk also contains antibodies like IgG and IgA which show antiviral activity 24, 26 . Therefore, these gave us confidence to continue to identify the anti-SARS-CoV-2 components in breastmilk and explore the underlying mechanisms. Here, we aimed to identify the protein components in human breastmilk by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) 27 and verify their antiviral activity using the SARS-CoV-2 pseudovirus and trVLP system in Huh7.5, Vero E6 and Caco-2-N cell lines, respectively 28 . We identified that LF, MUC1 and α-LA could inhibit SARS-CoV-2 and variants infection by blocking viral entry and post-entry replication, which shed light on the important role of LF, MUC1 and α-LA derived from human milk and provide the clues for the development of antiviral drug identification and design. To identify the effective factors of human skimmed milk influencing on SARS-CoV-2 infection, we performed mass spectrometry to identify the potential factors from the skimmed milk shown in Figure 1A . The skimmed milk was separated by cation exchange column, anion exchange column and size exclusion column in sequence. After these column separations, we got several fractions such as SP1, SP2, SP3, Q1 and Q2, which were subsequently collected for anti-SARS-CoV-2 analysis (Figure 1B to1D). We infected the Vero E6 cells with 650 TCID50/well of SASRS-CoV-2 pseudovirus and added same ratio of SP1, SP2, SP3, Q1 and Q2 into the media, the skimmed breastmilk (A17) was used as control. One day post infection, we found that SP2, SP3 and Q2 exhibited inhibitory effects to SARS-CoV-2 pseudovirus infection ( Figure 1E) . Then, we selected these fractions into tandem mass spectrometry (MS/MS) analysis and found that LF were the potential factors for SARS-CoV-2 inhibition ( Figure 1F and Figure S1 to S4). These data suggested that LF is the potential factor in skimmed milk inhibiting SARS-CoV-2 infection. As reported in our previous publication, LF might not be the effective factor alone in breastmilk and there might be other components involving the anti-SARS-CoV-2 infection 22 . Previous reports showed that some factors, such as MUC1, MUC4, lactadherin, α-LA, etc., have anti-microbe activity 23, 24 . To identify which of them could also inhibit SARS-CoV-2 infection, we mixed the MUC4, MUC1, α-LA, lactadherin and LF with 650 TCID50/well of SASRS-CoV-2 pseudovirus with luciferase expression, respectively. Then, the mixture was added to infect Vero E6 cell for 24h. MUC4 ( Figure 2A ) and lactadherin ( Figure 2D ) were showed no suppression to SARS-CoV-2 pseudovirus infection. Interesting, MUC1 (0.25 μg/ml, Figure 2B ), α-LA (0.25 mg/ml, Figure 2C ) and LF (0.25 mg/ml, Figure 2E ) showed dramatic inhibition of SARS-CoV-2 pseudovirus infection. In addition, we also used the GFP expressing SARS-CoV-2 pseudovirus to verify the inhibition activity of LF, MUC1 and α-LA in both Huh7.5 and Vero E6 cell lines. As shown in Figure 2F to 2H, all the LF, MUC1 and α-LA could suppress GFP expression, indicating that these factors in skimmed milk could inhibit SARS-CoV-2 infection. Thus, these three factors in breastmilk were validated to inhibit SARS-CoV-2 pseudovirus infection. Figure 3B) . Similarly, GFP expressing reflecting the viral replication revealed that both LF and MUC1 could significantly suppress SARS-CoV-2 replication with low GFP expression ( Figure 3C ). Although we could see the inhibition effect of α-LA on SARS-CoV-2 infection, the inhibitory ability is relatively lower, which still showed a few GFP expression even in concentration of 2 mg/ml. Consistent to the GFP results, western blot analysis of the cell lysates showed that MUC1 and LF could significantly suppress the S protein expression of SARS-CoV-2 ( Figure 3D) . The α-LA at high concentration of 2 mg/ml could also suppress SARS-CoV-2 infection. In addition, to explore whether the LF from different species possessed inhibitory effect on SARS-CoV-2 infection, we tested the inhibitory effect of recombinant human LF (rLF), human isolated LF (hLF), bovine LF (bLF) and goat LF (gLF) on SARS-CoV-2 infection. The skimmed milk (A17) was used as positive control. Firstly, we tested them in the SARS-CoV-2 pseudovirus system. We infected the Vero E6 cells with 650 TCID50/well of SASRS-CoV-2 pseudovirus and treated the cells with rLF, hLF, bLF, gLF and A17 for 24h. After treatment, luciferase assay showed that all of the LFs and A17 could significantly suppress SARS-CoV-2 pseudovirus infection, indicating that LF from bovine and goat could also inhibit SASR-CoV-2 infection ( Figure 4A ). Consistent to the pseudovirus system, trVLP system showed that all the LFs could also inhibit SARS-CoV-2 infection and replication in RT-qPCR analysis ( Figure 4B ) and immune microscopy analysis of GFP expression ( Figure 4C) . Western blot analysis of the cell lysates showed that rLF and bLF significantly suppressed SARS-CoV-2 S protein expression ( Figure 4D ). In addition, we found that gLF at high concentration of 2 mg/ml could be toxic to cell (Figure 4E) , while other LFs showed no toxic to cell. for 96 h, we found that gLF suppressed SARS-CoV-2 infection in a dose dependent manner. However, CCK8 analysis showed that gLF at high concentrations exhibited high cytotoxicity to Caco-2-N cells with CC50 of 5.64 mg/ml ( Figure 4F) . Thus, LF, MUC1 and α-LA from human breastmilk were confirmed to suppress SARS-CoV-2 infection and replication. In addition, LF from other species could also inhibit SARS- We performed a dose-response experiment with LF, MUC1 and α-LA to assess their suppression on SARS-CoV-2 infection and replication. Firstly, we explored them in the trVLP system infected Caco-2-N cells. The results confirmed that the inhibitory effects of MUC1, LF and α-LA on SARS-CoV-2 infection were dose dependent and the 50% effective concentration (EC50) for MUC1, LF and α-LA was as low as 0.04 μg/ml ( Figure 5A ), 0.08 mg/ml ( Figure 5B ) and 0.68 mg/ml (Figure 5C ), respectively. Surprisingly, the cytotoxic values evaluated by CCK-8 assay showed that MUC1, LF and α-LA have no cytotoxicity to the cells, even the highest concentration of MUC1(2 μg/ml), LF (2 mg/ml) and α-LA (2 mg/ml). Consistent to these results, we also tested them in the SARS-CoV-2 pseudovirus system infected Vero E6 cells. As shown in Figure S5A , the results confirmed the inhibitory effects of MUC1 on SARS-CoV-2 pseudovirus infection and exhibited that the EC50 for MUC1 was as low as 0.1 μg/ml. Similarly, the EC50 of LF and α-LA was as low as 0.1 mg/ml ( Figure S5B ) and 0.16mg/ml (Figure S5C ), respectively. When treating the cells with relative lower concentration of MUC1, LF and α-LA in the trVLP infected Caco-2N cells, we found that LF and MUC1 could significantly Figure 6B) . Similarly, the GFP expressing results showed that cells treated with these factors nearly expressed no GFP ( Figure 6C) . We also tested them in the pseudovirus system ( Figure S6A ). Consistent reductions in the luciferase expression were identified in the SARS-CoV-2 pseudovirus infected Vero E6 and Huh7.5 cells ( Figure S6B ). These data indicated that MUC1, LF and α-LA might attach the cells to block viral attachment and entry. Then, we also designed the post-infection experiment as shown in Figure 6D . In addition, we also performed the experiment to explore if MUC1, LF and α-LA suppress viral entry. We infected trVLP into the Caco-2-N cells and pseudovirus into the Huh7.5 and Vero E6 cells at 4℃ for 1h, respectively. Then, we washed the virus away and changed the media supplemented with these factors until the end point of infection. Interestingly, all these factors showed potent antiviral activity as evidence by the dramatic reduction of the SARS-CoV-2 RNA ( Figure 6G ) and luciferase activity levels ( Figure S6F) . These data suggested that MUC1, LF and α-LA suppress SARS-CoV-2 entry step of viral life cycle. To explore if MUC1, LF and α-LA interfere the interaction of ACE-2 and S protein, we performed the affinity assay. As shown in Figure 7A and 7B, LF and MUC1 didn't block the interaction of ACE-2 and S protein, indicating that LF and MUC1 suppress SARS-CoV-2 infection through other ways rather than the ACE-2target. Surprisingly, α-LA could interfere SARS-CoV-2 attachment to ACE-2, showing high absorbance after α-LA treatment at low concentration of 0.25 mg/ml ( Figure 7C) . Moreover, it was reported that LF inhibit SARS-CoV infection through the HSPG target 19 . We also performed the experiment to explore the possible same targetfor LF to inhibit SARS-CoV-2 infection. We treated the cells with LF combined with different concentrations of heparin. We found that the inhibitory effect of LF and MUC1 on SARS-CoV-2 decreased with dose-dependent of heparin combined treatment, to the deep when 10 U/ml of heparin was added in both Huh7.5 (Figure 7D and 7F) and Vero E6 (Figure 7E and 7G) cells. However, when increasing the heparin to 100 U/ml, we found that the inhibitory effect increased, indicating that heparin could interfere the inhibitory effect of LF on SARS-CoV-2 infection. To further confirm that LF and MUC1 interact SARS-CoV-2 attaching HSPG, we also performed new experiments in the trVLP infected Caco-2-N cells as designed in Figure S7A . as the results, we also found that MUC1 ( Figure S7B ) and LF ( Figure S7C ) rather than α-LA ( Figure S7D ) could interfere heparin's inhibition of SARS-CoV-2 infection. These data suggested that one way of LF suppressing SARS-CoV-2 infection is by inhibiting virus to attach to HSPG. To determine whether MUC1, LF and α-LA still inhibit SARS-CoV-2 variants, we That means, LF shows high concentration in breastmilk and effectively inhibits SARS-CoV-2 possible infection and transmission. This analysis suggested that breastmilk feeding may be safe for infants whose mother are infected with SARS-CoV-2, if the breastmilk was pumped into sterile containers and feed infants with isolation from their mother. However, it still needs further study to confirm the safety of breastfeeding to infants from SARS-CoV-2 infected mother. SARS-CoV-2 belongs to RNA virus and is easy to mutate during the infection, which resulting in viral escape from antibody neutralization, vaccination, and other drugs targeting the interaction between S protein and ACE-2 4 . Surprisingly, LF, MUC1 and α-LA still could inhibit the existed mutants such as B. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Kuanhui Xiang (kxiang@bjmu.edu.cn) All materials in this study are available from the Lead Contact with a completed Materials Transfer Agreement. The original sequencing datasets for trVLP of SARS-CoV-2 can be found on the The skimmed milk samples preparation was performed as previously described. Briefly, the milk samples were centrifuged for 15 min at 4,000 x g at 4°C and the lower aqueous phase was collected and used for further experiments and analysis 34 . The viral production was performed as previously described 28 SARS-CoV-2 pseudovirus infection was performed as described 34 . The cells were infected with viral inocula of 650 TCID50/well. One day post infection (1dpi), the cells were lysed and the luminescence was measured according to the manufacture's protocol. For trVLP, Caco-2-N cells were infected trVLP at multiplicity of infection (MOI) of 0.1 as described. Several days post-infection, the mRNA levels of trVLP and GAPDH were determined by RT-qPCR. Upon SDS-PAGE fractionation, the band of interest was excised and subjected to ingel trypsin digestion as previously described 27 acetonitrile, and 0.1% formic acid; B: 80% acetonitrile, 20% water, and 0.1% formic acid). The LC gradient started at 7% B for 3 min and then was linearly increased to 37% in 40 min. Next, the gradient was quickly ramped to 90% in 2 min and stayed there for the mass spectrometer for MS and MS/MS analyses in a data dependent acquisition mode. One full MS scan (m/z 400-1200) was acquired by the Orbitrap mass analyzer with R = 60,000 and simultaneously the ten most intense ions were selected for fragmentation under collisioninduced dissociation (CID). Dynamic exclusion was set with repeat duration of 30 s and exclusion duration of 12 s. Caco-2N cells were seed at the 24-well plates with 80,000 cells/well one day before the infection. LF (2 mg/ml), MUC1 (2 ug/ml) and α-LA (2 mg/ml) was mixed with trVLP (MOI=1) at 4℃ for 1h, respectively. The mixture was added into the cells and put at 4℃ for 2h to allow viral attachment to cells. After washing out of free virus, cell surface GX_P2V was extracted and quantified by RT-qPCR. For SARS-CoV-2 pseudovirus, Huh7.5 and Vero E6 cells were seed at the 96-well plates with 20,000 cells/well one day before infection. LF (2 mg/ml), MUC1 (2 ug/ml) and α-LA (2 mg/ml) was mixed with trVLP (MOI=1) at 4℃ for 1h, respectively. The mixture was added into the cells and put at 4℃ for 2h to allow viral attachment to cells. After washing out of free virus, the cells were incubated at 37℃ for 24 h. The luciferase assay was performed to detect SARS-CoV-2 pseudovirus infection. For SARS-CoV-2 pseudovirus, the method was described as previously described 34 . Caco-2-N cells were exposed to trVLP (MOI=1) at 4 ℃ for 1h. Then, the cells were washed with PBS for 3 times. LF (2 mg/ml), MUC1 (2 ug/ml) and α-LA (2 mg/ml) were added into the media and incubated at 37℃ for 1h to allow viral internalization into cells, respectively. The mRNA of trVLP was measured by RT-qPCR. GFP were detected by a fluorescence microscope (ECHO laboratories, USA). For SARS-CoV-2 pseudovirus, the method was described as previously described 34 . Caco-2-N cells were infected with trVLP and incubated at 37 ℃ for 1h. After washing out of the free viruses, the cells were cultured in the media containing LF (2 mg/ml), MUC1 (2 ug/ml) and α-LA (2 mg/ml), respectively. Different time point of postinfection, the mRNA of trVLP was measured by RT-qPCR. GFP were detected by a fluorescence microscope (ECHO laboratories, USA). For SARS-CoV-2 pseudovirus, the method was described as previously described 34 . The RNA extraction and quantification were performed as previously described 28 . Briefly, Total RNA of cells was isolated with the RNAprep pure Cell Culture/Bacterial total RNA extraction kit (Tiangen biotech. Co., China). The cDNA was synthesized by RevertAid first strand cDNA synthesis kit (Invitrogen, USA). The qPCR for SARS-CoV-2 RNA was performed as previously described. The qPCR primers for viral RNA were as follows: THU-2190 (5'-CGAAAGGTAAGATGGAGAGCC-3') and THU-2191 (5'-TGTTGACGTGCCTCTGATAAG-3'). GAPDH was used to normalize all the data 28 . Western blotting was performed as described previously 34 The influence of LF, MUC1 and α-LA on the affinity between ACE2 and SARS-CoV-2 S RBD was performed as previously described 34 Scale bar represents 100 μm. Data are presented as mean± SD and repeated at least three times (N = 3), **p < 0.01, ***p < 0.001, ****p<0.0001. (G) The viral RNA levels detected by RT-qPCR are suppressed by MUC1, α-LA, rLF, hLF, bLF and A17 during viral entry. The trVLP was mixed with Caco-2-N cells at 4 ℃ for 1 hour then discarded the supernatant and washed with PBS for three times. The cells were then added with fresh media with MUC1, α-LA, rLF, hLF, bLF and A17 treatment at 37 ℃ for 2 hours. 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MUC1 (2 ug/ml), α-LA (a-lactalbumin, 2 mg/ml), rLF (recombinant lactoferrin, 2 mg/ml), hLF (human lactoferrin, 2 mg/ml), bLF (bovine lactoferrin, 2 mg/ml) and A17 (skimmed milk, 2 mg/ml). Scale bar represents 100 μm. Data are presented as mean± SD and repeated at least three times LA interferes the affinity of spike protein and ACE-2, MUC1 and LF inhibits SARS-CoV-2 attaching to HSPG C) on the affinity between ACE2 and SARS-CoV-2 RBD. (D) and (E) inhibition of SARS-CoV-2 pseudovirus infection of Huh7.5 and Vero E6 cells were treated with heparin for 10 min at the dosage of 0.1 U/ml, 1 U/ml Then, 2 mg/ml LF was added to each group and incubated at 37℃ for 1h F) and (G) inhibition of SARS-CoV-2 pseudovirus infection of Huh7.5 and Vero E6 cells were treated with heparin for 10 min at the dosage of 0.1 U/ml, 1 U/ml 2 mg/ml LF was added to each group and incubated at 37℃ for 1h. The luciferase assay was performed to detect SARS-CoV-2 pseudovirus infection. Data are presented as mean± SD and repeated at least three times LA suppress different SARS-CoV-2 variants infection and replication in Caco-2-N cells. (A) Schematic illustration of the treatment experiment of MUC1, LF and α-LA for SARS-CoV-2 variants infection. The caco-2-N cells were seed in the six well plates and infected with SARS-CoV-2 variants of B The cells were treated with MUC1, rLF and α-LA, respectively during the infection. The PBS and the skimmed milk of A17 were used as negative and positive control, respectively. Fortyeight hours post-infection, the cells were harvested and tested. The inhibition analysis of MUC1, rLF and α-LA to SARS-CoV-2 variants of (B) B.1.1.7 (alpha), (C) B.1.351 (beta), (D) P.1 (gamma) and (E) B.1.617.1 (kappa) reflected by intracellular viral RNA were determined by RT-qPCR. Data are presented as mean± SD and repeated at least three times