Severe acute respiratory coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The frequent appearance of variants requires adjunct strategies to combat SARS-CoV-2 infections. Lactoferrin, a naturally occurring glycoprotein, exhibits promising antiviral properties. Here, we investigated the efficacy of bovine LF (bLF) against SARS-CoV-2 infection. SARS-CoV-2 pseudoviruses bearing the original Wuhan (wild-type), D614G mutant, or Omicron variant spike proteins were used to examine the effects of bLF on viral entry into host cells. We found that although bLF did not suppress entry of wild-type pseudoviruses into cells expressing both human angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), it did suppress entry of Omicron pseudoviruses into the cells. The results of this in vitro study highlight the spike protein-dependent effects of bLF on suppression of pseudovirus entry into host cells. Future clinical trials investigating the efficacy of bLF against SARS-CoV-2 infection should consider the differential impact of virus variants.

# Introduction

Severe acute respiratory coronavirus 2 (SARS-CoV-2) is a positive single-stranded, enveloped RNA virus that causes coronavirus disease 2019 (COVID-19) (Huang et al., 2020; Kawasuji et al., 2022). Since the onset of the COVID-19 epidemic in 2019, SARS-CoV-2 infection has resulted in approximately 7 million deaths worldwide (https://covid19.who.int/).

Similar to other RNA viruses, SARS-CoV-2 mutates rapidly, leading to emergence of new variants (Khatri et al., 2023). The first reported variant, harbouring missense mutation D614G in the spike (S) protein, was identified in February 2020 (Yurkovetskiy et al., 2020). The envelope S glycoprotein mediates entry of viruses into host cells, making them crucial determinants of cell tropism and pathogenesis (Hulswit, De Haan, & Bosch, 2016). Notably, several significant SARS-CoV-2 variants have emerged worldwide, including the Alpha variant in the United Kingdom, the Beta variant in South Africa, and the Gamma variant in Brazil (Rajah et al., 2021). Some mutations in these variants confer the ability to evade antibody therapy, enhance S protein binding to the human angiotensin-converting enzyme 2 (ACE2) receptor, and increase transmissibility (Rajah et al., 2021). The most recent SARS-CoV-2 variant, Omicron, emerged in South Africa at the end of 2021. To date, this variant harbours 37 mutations in the S protein gene and has developed several lineages, including BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5. Since early August 2022, the BA.5 lineage has become the dominant variant worldwide. The Omicron variant exhibits high transmissibility, milder symptoms, and the ability to evade vaccine-induced immune responses, which are crucial for controlling COVID-19. Furthermore, the frequent emergence of SARS-CoV-2 variants requires additional approaches to control infections (Hewins et al., 2022; Hui et al., 2022; Li et al., 2023; Shrestha, Foster, Rawlinson, Tedla, & Bull, 2022).

Lactoferrin (LF) is a 70–80 kDa iron-binding glycoprotein synthesised by neutrophils and exocrine glands. It is present in breast milk and other body secretions (Ikeda et al., 2000; Redwan, Uversky, El-Fakharany, & Al-Mehdar, 2014). Human LF comprises 691 amino acids, whereas bovine LF (bLF) comprises 689 amino acids; the two proteins show 69% sequence homology (Kell, Heyden, & Pretorius, 2020; Wakabayashi, Oda, Yamauchi, & Abe, 2014). LF is an important protein involved in innate immunity. Considering its broad-spectrum antiviral efficacy, safety, minimal side effects, and commercial availability, bLF is a promising candidate prophylactic or post-exposure treatment for SARS-CoV-2 infection (Kobayashi-Sakamoto et al., 2022, 2023).

Previous studies demonstrate that binding of SARS-CoV-2 to ACE2 is facilitated by heparan sulfate proteoglycans (HSPG), which acts as an attachment factor on host cell membranes (Schuurs et al., 2021). HSPG binds directly to the S protein of SARS-CoV-2, thereby promoting viral adhesion to the host cell surface and facilitating viral entry (Bermejo-Jambrina et al., 2021). Studies show that bLF prevents virus attachment to HSPG by binding to the latter directly (Bermejo-Jambrina et al., 2021; Hu, Meng, Zhang, Xiang, & Wang, 2021; Lang et al., 2011).

Several clinical trials have investigated the efficacy of bLF against SARS-CoV-2 (Einerhand et al., 2022). Compared with the control group, the LF-treated group exhibited SARS-CoV-2 infection of shorter duration, as measured by detection of fewer viral particles and less severe symptoms (Rosa et al., 2021). Patients infected with SARS-CoV-2 who were treated with liposomal bLF recovered faster than patients treated with conventional strategies, or those left untreated (Campione et al., 2021). However, large scale clinical trials have not yielded favourable results (Algahtani et al., 2021; Matino et al., 2023; Navarro et al., 2023).

Here, we aimed to assess the efficacy of bLF against SARS-CoV-2 infection. Specifically, we assessed the inhibitory effect of bLF on pseudoviruses carrying S proteins from the original Wuhan strain (wild-type), the D614G mutant, and the Omicron variant (BA.1 and BA.5).

# Materials and methods

## Reagents

African green monkey kidney epithelial (Vero E6) cells (ATCC CRL-1586) were purchased from ATCC, and VeroE6 cells expressing TMPRSS2 (VeroE6-TMPRSS2) (JCRB1819) were purchased from the Japanese Collection of Research Bioresources Cell Bank (National Institute of Biomedical Innovation, Health, and Nutrition, Osaka, Japan). Human A549-ACE2-TMPRSS2 cells (human ACE2-and TMPRSS2-expressing A549 cells) and A549-ACE2 cells (human ACE2-expressing A549 cells) generated from human lung A549 cells, as well as puromycin and hygromycin, were obtained from InvivoGen (San Diego, CA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Foetal bovine serum (FBS) was acquired from HyClone (Logan, UT, USA). Penicillin–streptomycin and a High-Capacity cDNA Reverse Transcription Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). GIT and bovine lactoferrin were obtained from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). Isogen II was purchased from Nippon Gene (Tokyo, Japan). Passive Lysis Buffer, a Luciferase Assay System, and GoTaq qPCR Master Mix were acquired from Promega Corporation (Madison, WI, USA). Camostat mesylate and E64d were obtained from Selleck Chemicals (Houston, TX, USA).

## Cell lines

VeroE6 and VeroE6-TMPRSS2 cells were cultured in low-glucose DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin–streptomycin.

Human A549-ACE2-TMPRSS2 and A549-ACE2 cells were maintained in high-glucose DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin–streptomycin, and 0.5 puromycin. A549-ACE2-TMPRSS2 cells were additionally supplemented with 300 μg mL−1 hygromycin. All cell lines were cultured at 37 °C in a 5% CO2 atmosphere.

## SARS-CoV-2 pseudoviruses

Pseudotyped vesicular stomatitis viruses (VSVs) encoding the luciferase gene and bearing the SARS-CoV-2 S protein of the original Wuhan (wild-type), that of variants D614G, Omicron BA.1, and Omicron BA.5, or the VSV-G glycoprotein (VSV-G) were generated as previously described (Kandeel et al., 2021; Ozawa et al., 2022; Tani et al., 2021; Yamada et al., 2022). To increase expression of the S protein on the cell surface, 19 amino acids were deleted from its C-terminus (Fukushi et al., 2005; Kawase, Shirato, Matsuyama, & Taguchi, 2009). The expression plasmids used for the truncated S protein were pCAG-SARS-CoV-2 S, pCAGG-pm3-SARS2-Shu-d19-B1.1.529.1 (Omicron BA.1-derived variant), and pCAGG-pm3-SARS2-Shu-d19-B1.1.529.5 (Omicron BA.5-derived variant) (Itamochi et al., 2023; Tani et al., 2021). VSVs bearing envelope (G) (VSV-G) were generated as a control. VSVs were stored at −80 °C until required.

## Infection with pseudoviruses

Cells were seeded in 48-well plates at a density of 8 × 104 cells per well and then cultured until the desired confluency was reached. Subsequently, the medium was replaced with serum- and antibiotic-free medium containing equal volumes of GIT and DMEM. After 4 h of incubation, the medium was replaced with fresh medium containing the specified concentrations of bLF. Following an additional 4 h of incubation, the cells were infected with the indicated VSVs for 24 h, and then lysed with Passive Lysis Buffer. Luciferase activity was measured by a Luciferase Assay System and a TriStar2 LB 942 Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany).

To examine the role of TMPRSS2 and cathepsins in bLF-mediated suppression of viral entry, cells were pretreated for 1 h prior to VSV infection with the TMPRSS2 inhibitor, camostat mesylate, and the cathepsin inhibitor, E64d. Cells used as the vehicle control group were treated with DMSO at a concentration equivalent to that of the inhibitors.

## Reverse transcription-quantitative polymerase chain reaction

Total RNA was extracted using Isogen II. Next, cDNA was synthesised using the High-Capacity cDNA Reverse Transcription Kit and amplified using GoTaq qPCR Master Mix and the Dice Real-Time PCR System (TP-951; Takara, Shiga, Japan). β-Actin (ACTB) was used as the internal control.

The following primers were used: IFNL1, 5′- GGTGACTTTGGTGCTAGGCT-3 (forward) and 5′-TGAGTGACTCTTCCAAGGCG-3′ (reverse); IFNL2 5′-GACAAGACCCAAACAGACCCT-3′ (forward) and 5′-GTCCCCAGTCATGTCTAGTTTC-3 (reverse); IFLB 5′-CTTTGCTCTGGCACAACAGG-3′ (forward) and 5′-GTGGAGAAGCACAACAGGAGA-3′ (reverse); and ACTB 5′-TGGCACCCAGCACAATGAA-3′ (forward) and 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ (reverse).

## Statistical analysis

Statistical analyses were performed using GraphPad Prism, version 9 (GraphPad Software Inc., La Jolla, CA, USA). Student's t-test was used to assess the statistical significance of differences between groups, and a p-value <0.05 was considered significant. Data are expressed as the mean of three independent experiments performed as three or four biological replicates (n). The value for each biological replicate is represented by a circle and the standard deviations are represented by error bars.

# Results and discussion

## bLF suppresses entry of SARS-CoV-2 Wuhan (wild-type) pseudoviruses into ACE2-expressing host cells but not TMPRSS-expressing cells

In this study we investigated the effects of both bLF and TMPRSS2 on entry of VSV pseudoviruses (VSVpv) and SARS-CoV-2 Wuhan (wild-type) pseudoviruses (CoV-2-Wtpv) into A549-ACE2, A549-ACE2-TMPRSS2, VeroE6, and VeroE6-TMPRSS2 cells. bLF-pretreated cells were infected with VSVpv or CoV-2-Wtpv, and the infection levels were compared in a luciferase assay (Fig. 1
                        A). The results showed that bLF suppressed entry of VSVpv and CoV-2-Wtpv into A549-ACE2 and VeroE6 cells (Fig. 1B and C). However, although bLF suppressed entry of VSVpv into A549-ACE-2-TMPRSS2 and VeroE6-TMPRSS2 cells, it did not suppress entry of CoV-2-Wtpv (Fig. 1D and E).

## Inhibition of TMPRSS2 activity promotes bLF-mediated suppression of CoV-2-Wtpv entry

Next, we examined the impact of TMPRSS2 activity on the inhibitory effect of bLF on CoV-2-Wtpv entry. Cells pretreated with camostat mesylate, a TMPRSS2 inhibitor, were infected with CoV-2-Wtpv or VSVpv (Fig. 2
                        A). Inhibition of TMPRSS2 activity in host cells by camostat mesylate increased bLF-mediated suppression of CoV-2-Wtpv entry (Fig. 2B and C). By contrast, pretreatment with camostat mesylate had no significant impact on entry of VSVpv, which occurred independently of TMPRSS2 activity (Fig. 2B). The SARS-CoV-2 Wuhan strain, which emerged in 2019, infects cells via two mechanisms. Upon binding to ACE2 expressed by host cells, SARS-CoV-2 relies on TMPRSS2 to enter host cells; however, in the absence of TMPRSS2, SARS-CoV-2 binds to ACE2 and is taken up by the cells through endocytosis facilitated by cathepsins (Hoffmann et al., 2020; Koch et al., 2021; Pišlar et al., 2020; Scudellari, 2021). Our findings demonstrate that inhibiting TMPRSS2 activity restores bLF-mediated suppression of CoV-2-Wtpv infection, suggesting that bLF may interfere with cathepsin-assisted entry into host cells.

## bLF suppresses entry of Omicron variant pseudovirus into ACE2-and TMPRSS2-expressing host cells to a greater extent than that of CoV-2-D614Gpv

Next, we investigated whether bLF-mediated suppression of viral entry into A549-ACE2-TMPRSS2 cells differs among the SARS-CoV-2 variant pseudoviruses (Fig. 3
                        A). Cells were pretreated with bLF and then infected with SARS-CoV-2 D614G mutant pseudovirus (CoV-2-D614Gpv) or the Omicron subvariant pseudoviruses (CoV-2-BA.1pv and CoV-2-BA.5pv). To examine the relationship between the inhibitory effects of bLF and host proteases, cells were pretreated with camostat mesylate (a TMPRSS2 inhibitor) or E64d (a cathepsin inhibitor) prior to infection with pseudoviruses.

The results showed that bLF suppressed entry of CoV-2-D614Gpv into A549-ACE2-TMPRSS2 cells by 22% (Fig. 3B). Camostat mesylate increased bLF-mediated inhibition of CoV-2-D614Gpv entry (to 44%), whereas E64d treatment had no significant effect (Fig. 3B). Additionally, bLF suppressed entry of both CoV-2-BA.1pv (by 64%) and CoV-2-BA.5pv (by 60%) (Fig. 3C and D). Furthermore, treatment with camostat mesylate slightly enhanced bLF-mediated suppression of CoV-2-BA.1pv (72%) and CoV-2 BA.5pv (69%) entry (Fig. 3C and D). By contrast, E64d significantly inhibited entry of CoV-2-BA.1pv and CoV-2-BA.5pv into both bLF-treated and control cells, with no significant difference between the two groups (Fig. 3C and D). These findings suggest that bLF effectively inhibits entry of the CoV-2-D614Gpv or Omicron variants through the cathepsin-mediated infection route, but that it is not as effective against the TMPRSS2-mediated entry route.

Next, we focused on the effects of protease inhibitors. Pretreatment with camostat mesylate suppressed entry of CoV-2-D614Gpv into A549-ACE2-TMPRSS2 by 63% (Fig. 3B), whereas E64d pretreatment had no significant impact (Fig. 3B). By contrast, camostat mesylate pretreatment suppressed entry of CoV-2-BA.1pv by 28%, whereas E64d suppressed it by 87% (Fig. 3C). Regarding CoV-2-BA.5pv, pretreatment with camostat mesylate had no significant effect on viral entry, whereas E64d suppressed viral entry by 86% (Fig. 3D). These findings suggest that CoV-2-D614Gpv preferentially uses the TMPRSS2-dependent pathway for viral entry, whereas Omicron variants BA.1 and BA.5 preferentially use the cathepsin-dependent pathway.

Previously, we reported that bLF inhibits entry of CoV-2-Wtpv into Caco2, a small intestine-like cell line that co-expresses TMPRSS2 and ACE2 (Kobayashi-Sakamoto et al., 2022). Consistent with our findings, other studies reported similar results (Mirabelli et al., 2021; Salaris et al., 2021; Yazawa et al., 2023). Therefore, expression of ACE2 and TMPRSS2 in host cells might influence the effectiveness of bLF directly, which requires further research.

Wild-type SARS-CoV-2 and the D614G mutant rely on TMPRSS2 for entry into host cells. By contrast, the Omicron variants, namely, BA.1 and BA.5, seem to be less dependent on TMPRSS2 for viral entry (Khatri et al., 2023). As shown in Fig. 3, while CoV-2-D614Gpv enters the cell through a TMPRSS2-dependent pathway, the Omicron pseudoviruses preferentially use the cathepsin-dependent pathway. These findings are consistent with previous studies (Khatri et al., 2023; Meng et al., 2022; Padmanabhan & Dixit, 2023; Tang et al., 2021). Additionally, our results suggest that bLF suppresses cathepsin-dependent entry of all SARS-CoV-2 variants investigated in this study, suggesting that the efficacy of bLF varies depending on the host proteases involved in viral entry.

While bLF was unable to suppress entry of CoV-2-Wtpv into TMPRSS2-expressing host cells, it did prevent entry of CoV-2-D614Gpv into host cells. However, the most notable effect of bLF was to suppress entry of Omicron variant pseudoviruses into host cells. The D614G mutation enhances the infectivity of SARS-CoV-2 and promotes entry into host cells via the cathepsin pathway (Laporte et al., 2021; Padmanabhan & Dixit, 2023; Tang et al., 2021). Therefore, bLF-mediated suppression of CoV-2-D614G entry into TMPRSS2-expressing host cells might be due to selective use of host cell proteases.

The mechanism underlying the selective inhibition of the cathepsin-mediated entry pathway by bLF remains unknown. Reports suggest that bLF inhibits entry of SARS-CoV and SARS-CoV-2 by binding to HSPGs on host cells (Bermejo-Jambrina et al., 2021; Hu et al., 2021). Additional studies are required to determine whether this interaction is involved in the bLF-mediated inhibition of viral entry. Previous studies reported that bLF inhibits cathepsin activity (Ohashi et al., 2003; Patil, Chen, Fogliano, & Madadlou, 2023). Collectively, these findings suggest that bLF suppresses viral entry by directly inhibiting cathepsin activity in the host cell. However, it is unclear whether exogenous bLF inhibits activity of intracellular cathepsins in host cells.

## bLF modulates expression of mRNA encoding interferon-λ (IFNλ) and IFNβ induced by SARS-CoV-2 pseudovirus infection of host cells

Finally, we asked whether bLF affects expression of IFNs by TMPRSS2-expressing host cells following infection with SARS-CoV-2 pseudoviruses (Fig. 4
                        A). After a 12 h infection with CoV-2-D614Gpv, bLF treatment suppressed expression of mRNA encoding IFNλ2 in A549-ACE2-TMPRSS2 cells (Fig. 4C). Additionally, bLF suppressed expression of mRNAs encoding IFNλ1, IFNλ2, and IFNβ following CoV-2-BA.5pv infection (Fig. 4B–D). The effect of bLF on IFN mRNA expression disappeared at 24 h post-infection (Fig. 4B–D). These results suggest that the impact of bLF on the IFN response is greater in cells infected by SARS-CoV-2 Omicron variants than in cells infected by the CoV-2-D614G mutants. Furthermore, bLF appears to affect IFN production by host cells at the early stage of SARS-CoV-2 infection, irrespective of the SARS-CoV-2 variant; indeed, this effect lessened as the infection process progressed.

SARS-CoV-2 infections are associated with severe clinical manifestations, resulting in part from a cytokine storm (excessive release of cytokines triggered by the innate immune system upon infection) (Zimecki, Actor, & Kruzel, 2021). Patients with severe SARS-CoV-2 infection exhibit elevated serum concentrations of interleukin (IL)-1α, IL-1B, IFNα, and IFN-λ (Perlman, 2020). In our study, we observed that bLF treatment modulated expression of mRNA encoding IFNs during the early stages of infection by SARS-CoV-2 variants (Fig. 4). This was more prominent for CoV-2-BA.5pv infection than for CoV-2-D614Gpv infection (Fig. 4). In general, Omicron variants are slow to induce immune responses; therefore, the immune system is unable to effectively combat the virus at the early stages of infection (Hu et al., 2022; Li et al., 2023; Shrestha et al., 2022). Further studies are required to better understand the in vivo efficacy of bLF-induced down-regulation of IFN production.

# Conclusion

To examine the efficacy of bLF against SARS-CoV-2, we investigated its impact on entry of different SARS-CoV-2 pseudoviruses into host cells. Specifically, we investigated entry of SARS-CoV-2 pseudoviruses bearing the Wuhan (wild-type), D614G mutant, and Omicron (BA.1, BA.5) variant S proteins. The findings demonstrate that bLF suppresses entry of CoV-2-Wtpv into ACE2-expressing cells but not into TMPRSS2-expressing cells (Fig. 1B and C). By contrast, bLF did not prevent entry of CoV-2-Wtpv into cells expressing both ACE2 and TMPRSS2 (Figs. 1D,E, 2B,C). Additionally, inhibition of TMPRSS2 activity restored bLF-mediated suppression of CoV-2-Wtpv entry (Fig. 2B and C). These findings suggest that bLF does not suppress entry of CoV-2-Wtpv through the TMPRSS2-mediated route.

Continued evolution of SARS-CoV-2 remains an important global public health concern. Despite the success of vaccines in various countries, access is still limited. bLF is an inexpensive and harmless nutrient found in food products, which has evident beneficial effects on SARS-CoV-2 infection. Our findings suggest that bLF-mediated suppression of viral entry differs among SARS-CoV-2 variants, highlighting the importance of considering specific viral variants in future clinical trials designed to assess the efficacy of bLF for SARS-CoV-2 infections.

# Author contributions

Conceptualization, M. K-S., H.T., and T. S.; Methodology, M. K-S., H.T., and T. S.; Investigation, M. K-S.; Writing – Original Draft Preparation, M. K-S.; Data Curation, H.T. and T. S.; Supervision, H.T., T.M., Y.K., and K.H.; Project Administration, H.T., T.M., Y.K., and K.H.; Funding Acquisition, M. K-S., H.T., T.M., Y.K., and K.H.; Writing – Review & Editing, H.T., T.M., Y.K., and K.H. All authors read and agreed with the published version of the manuscript.

# Data availability

All data generated and analysed during this study are available from the corresponding author upon reasonable request.

This research was supported by JSPS KAKENHI (Grant Number: 19K10074, 22K10344 and 22K11783), and a Grant-in-Aid for Scientific Research from the Ohu University School of Dentistry, including the President's discretionary expense (Grant Number Gakusaihi 2).