Antiviral Properties of Lactoferrin

Scientific Review on Antiviral properties of milk proteins and peptides

Scientific Review on Antiviral properties of milk proteins and peptides

Y. Pana, A. Leeb, J. Wanb, M.J. Coventryb, W.P. Michalskic, B. Shiellc, H. Roginskia,√É School of Agriculture and Food Systems, The University of Melbourne, Sneydes Rd., Werribee, VIC 3030, Australia b Microbiology and Biotechnology, Food Science Australia, Private Bag 16, Werribee, VIC 3030, Australia c Protein Biochemistry and Proteomics Group, Australian Animal Health Laboratory, CSIRO Livestock Industries, Private bag 24, East Geelong, VIC 3220, Australia

A substantial body of evidence, primarily from in vitro studies, suggests that some milk proteins interfere with viral infections.Lactoferrin (LF) has been the protein most comprehensively studied for its antiviral effects. Interference with viral infections is primarily based on adsorption of LF to receptors on the host cell‚ surface or on binding to viral particles, both enveloped and non-enveloped. In either mechanism, viral particles are prevented from attaching to host cells. Electrostatic attraction seems to play an important role in both mechanisms. In general, bovine LF is more effective against viral infections than human LF. Apo-LF is less effective than the ironsaturated LF. Antiviral effects of lactoferricin and other peptides liberated from LF are weaker than those of intact LF. Proteins other than LF, such as lactadherin, and peptides such as glycomacropeptide, also interfere with infection by some viruses. Chemical modifications of milk proteins that lead to changes in charges on proteins, and in charge distribution, enhance their effects against certain viruses.


With the progress in the knowledge of the composition and role of milk components it became apparent during the first half of the 20th Century that some milk components possessed biological properties beyond their nutritional significance. One of the earliest observations concerned the link between peroxidative activity of milk and its bactericidal properties (Hansen, 1924). Belding, Klebanoff, and Ray (1970) reported the virucidal effect of the lactoperoxidase system against poliovirus (PV) and vaccinia virus. A few years later, Fieldsteel (1974) observed that non-specific antiviral substances in the cream fraction of human milk were effective against arbovirus and murine leukaemia virus. The effectiveness of proteins in human and bovine milk against arbovirus, rhinovirus and influenza viruses was demonstrated by Matthews, Nair, Lawrence, and Tyrrel (1976). In 1987, lactoferrin (LF) was first reported to exert a protective effect in mice infected with Friend leukaemia virus (FLV) (Lu et al., 1987).

Since then, the antiviral effects of LF have been widely studied.
Results of these studies have been reviewed by Valenti et al. (1998), van Hooijdonk, Kussendrager, and Steijns (2000), Van Der Strate, Beljaars, Molema, Harmsen, and Meijer (2001), and Seganti et al. (2004). The present review focuses on the antiviral properties of milk proteins including LF, peptides derived from these proteins and chemically modified derivatives of milk proteins and peptides. 2. Antiviral properties of LF LF has been reported to interfere with the action of a number of enveloped viruses such asherpes simplex types 1 and 2, human cytomegalovirus, human immunodeficiency virus (HIV), hepatitis B, C and G viruses, human papillomavirus (HPV) and alphavirus.

In addition, LF is effective in vitro against several non-enveloped viruses like rotavirus, enterovirus, PV, adenovirus and feline calicivirus (FCV).
Human immunodeficiency virus In the early 1990s, it was reported that milk, a source of highly positively charged macromolecules, inhibited the binding of HIV type 1 (HIV-1) to CD4 receptor (Newburg, Viscidi, Ruff, & Yolken, 1992). The activity of LF against HIV-1 has been widely studied since the first report of its activity against this virus (Harmsen et al., 1995). Bovine LF (bLF) and human LF (hLF) were potent inhibitors of HIV-1-infection in vitro (Harmsen et al., 1995; Puddu et al., 1998; Swart, Harmsen, et al., 1996; Swart, Harmsen, et al., 1999), with bLF being a more potent agent against HIV-1 than hLF (Berkhout, Floris, Recio, & Visser, 2004), and there was little difference in the inhibitory activity of bLF from milk, colostrum or serum (Berkhout et al., 2004). The action of LF against HIV-1 takes place in an early phase of infection, probably during adsorption of the virus to target cells (Harmsen et al., 1995; Puddu et al., 1998; Van Der Strate et al., 2001). Electrostatic interactions seem to play an important role in the contact between the HIV-1 virion particles and the host cells (Roderiquez et al., 1995). It has been proposed that a large number of negatively charged compounds may specifically interact with the positively charged V3 loop of gp120 (the envelope protein) of HIV-1 and block the virus adsorption (Berkhout et al., 2002).

Herpes simplex virus – hLF and bLF interfere with the infection by both herpes simplex virus types 1 and 2 (HSV-1 and -2) in vitro (Fujihara & Hayashi, 1995; Hammer, Haaheim & Gutteberg, 2000; Hasegawa, Motsuchi, Tanaka, & Dosako, 1994; Marchetti et al., 1996, 1998). In addition, LF and lactoferricin (LFcin, see Section 3) have a synergistic antiviral effect in combination with an antiviral drug, acyclovir (Andersen, Jenssen & Gutteberg, 2003). Similar to the action against HIV, the antiviral effect of LF occurs in the early phase of HSV-infection, with LF inhibiting the adsorption of virus to the target cells by binding to virus particles (Hasegawa et al., 1994; Marchetti et al., 1996). In addition, bLF inhibits the in vitro infection by feline herpesvirus (FHV-1) prior to and during viral adsorption (Beaumont, Maggs, & Clarke, 2003). Bovine LF and its apo- and holo- forms, as well as hLF, were effective against canine herpesvirus (CHV) with the same mechanism as that of HSV (Tanaka et al., 2003). The net positive charge, and other features such as hydrophobicity, molecular size and spatial distribution of charged and lipophilic amino acids all seem to be important factors for the activity against HSV (Jenssen, Andersen, Uhlin-Hansen, Gutteberg, & Rekdal, 2004).

Cytomegalovirus – The effect of LF against cytomegalovirus (CMV) has been reported by several research groups (Clarke & May, 2000; Harmsen et al., 1995; Hasegawa et al., 1994; Shimizu et al., 1996; Swart, Harmsen, et al., 1999). According to these reports, LF interferes with the entry of virus into the target cell and inhibits the expression of early and late human CMV (HCMV; Andersen, Osbakk, Vorland, Traavik, & Gutteberg, 2001) and produces a synergistic effect against HCMV in combination with some antiviral drugs such as cidofovir (Van Der Strate et al., 2003). The N-terminal region of LF is essential for its activity against CMV (Swart et al., 1998; Swart, Harmsen, et al., 1999). Shimizu et al. (1996) reported that LF had anti-CMV activity in vivo in a mouse model by upregulation of natural killer cells. In contrast, Beljaars et al. (2004) indicated that LF exerted its antiviral effects by interfering with the virus entry into the cell rather than through stimulation of the immune system. The protease in the cell culture was thought to degrade cell-bound LF as the antiHCMV activity of LF completely disappeared after 60 min incubation (Harmsen et al., 1995). Desialylation of LF did not affect its activity against HCMV, indicating that protein moiety interactions are important during inhibition (Harmsen et al., 1995).

Hepatitis virus – Ikeda et al. (1998) developed an in vitro culture system for hepatitis C virus (HCV) replication and observed that LF inhibited this process. In a separate study, LF was also shown to be active against hepatitis G virus (HGV) as well as against HCV (Ikeda et al., 2000). LF seemed to directly interact with viral particles and prevent the viral infection by binding to the HCV envelope proteins E1 and E2 (Yi, Kaneko, Yu, & Murakami, 1997). The study of Nozaki, Tanaka, Naganuma, and Kato (2002) confirmed this finding and proved that bLF was effective in some patients with chronic hepatitis, after an 8-week course of the oral administration of the protein. Ishii et al. (2003) reported that oral administration of bLF may be effective in a clinical application against HCV in combination with interferon. In the action against hepatitis B virus (HBV), bLF and hLF appear to interact with target cells and prevent the viral infection (Hara et al., 2002). 2.1.5. Other enveloped viruses A few decades ago, breastfeeding was found to have a protective effect against respiratory syncytial virus (RSV) infection (Downham, Scott, Sims, Webb, & Gardner, 1976; Pullan et al., 1980). Later, Grover, Giouzeppos, Schnagl, and May (1997) discovered that hLF plays a critical role in preventing RSV infection. Very little is known, however, about the mode of action of LF against RSV. Human LF is also effective against FLV (Chen, Lu, & Broxmeyer, 1987; Lu et al., 1987; Vorland, 1999), a murine retrovirus which causes erythroleukemia in mice (Lu et al., 1987). Bovine LF also has an effect in vitro against hantavirus, a rodentborne zoonotic virus, by interfering with adsorption of the virus to cells. The antiviral drug, ribavirin, had a synergistic effect with LF on the replication of hantavirus (Murphy et al., 2000). Human serum albumin (HSA), positively charged by amination, and hLF had an effect against alphaviruses, indicating that the positive charge in LF plays an important role in its antiviral activity (Waarts et al., 2005).

Moreover, LF interfered with internalisation of HPV into the host cells by blocking the process of HPV uptake in the early infection phase. HPV is the cause of genital warts and a prerequisite for cervical cancer; it binds to the cell surface through the heparan sulphate receptor. Bovine LF is a more potent inhibitor of HPV entry than hLF (Drobni, Naslund, & Evander, 2004). It is clear that LF can inhibit viral infection by interfering with virus receptor interactions on the cell surface.

Non-enveloped viruses – LF is also active against several non-enveloped viruses including rotavirus, PV, FCV and adenovirus.

Rotavirus – Rotavirus infections are the most frequent cause of nonbacterial gastroenteritis in neonates and young children in the world, leading to approximately 1 million deaths world-wide every year (Blacklow & Greenberg, 1991). LF was found to be active against a simian rotavirus SA11 in vitro and the apo-LF was as potent against this rotavirus as the iron-saturated holo-LF form (Superti, Ammendolia, Valenti, & Seganti, 1997). Superti et al. (2001) demonstrated that the manganese or zinc-saturated bLF had a slightly lower anti-rotavirus effect than apo-bLF and ironsaturated bLF. Desialylation enhanced antiviral potency of bLF (Superti et al., 2001). This group also demonstrated that by binding to the viral particles bLF prevented rotavirus attachment to intestinal cells (Superti et al., 1997, 2001). In contrast, in the study of hLF by Grover et al. (1997), no effect against rotavirus was observed.

Poliovirus – PV is the cause of poliomyelitis, which can lead to paralysis of limbs. LF was effective against the PV replication in a dose-dependent manner (Marchetti et al., 1999). Apo- and native LF, as well as LF saturated with ferric, manganese or zinc ions prevented viral adhesion to Vero cells. However, only zinc-saturated LF strongly inhibited viral infection at the stage of virus internalisation into the host cells (Marchetti et al., 1999). It appears that LF binds to Vero cells and blocks the attachment of virus to the cells (Marchetti et al., 1999). The anti-PV effect of bLF was also observed in studies of McCann, Lee, Wan, Roginski, & Coventry (2003) where LF was detected on the surface of host cells.

Feline calicivirus – It has been estimated that between 30% and 40% of food-borne illnesses are caused by viral agents (Fleet, Heiskanen, Reid, & Buckle, 2000) and up to 70% of viral gastroenteritis cases are thought to be caused by Noroviruses (NV; formerly known as Norwalk-like viruses: Hale, 1999). However, the studies of NV have been hampered by the failure to grow the virus in tissue culture and the absence of an animal model (Jiang, Wang, Wang, & Estes, 1993). FCV is culturable and shares a number of biochemical properties, similar genomic organisation and primary sequences with NV and therefore has been used as a virus surrogate to study NV, (Doultree, Druce, Birch, Bowden, & Marshall, 1999; Slomka & Appleton, 1998). McCann et al. (2003) reported that bLF was active against FCV when it was added to cells prior to or together with the viral inoculation, indicating that LF interfered with the FCV infection at an attachment stage. Kreutz, Seal, & Mengeling (1994) reported that FCV interaction with cells is receptor mediated.

Other non-enveloped viruses – Adenovirus causes acute respiratory diseases, epidemic conjunctivitis and acute gastroenteritis among infants and young children (Di Biase et al., 2003). Apo- and native bLF and hLF are effective against adenoviruses in a dosedependent manner; their action takes place at an early phase of viral infection (Arnold et al., 2002). LF seems to prevent adenovirus from attachment to cell membranes through competition for common glycosaminoglycan receptors and through a specific interaction with viral structural polypeptides (Dechecchi et al., 2001; Dechecchi, Tamanini, Bonizzato, & Cabrini, 2000) and the N-lobe of bLF is particularly important in its antiviral action (Di Biase et al., 2003). In the study of Tinari, Pietrantoni, Ammendolia, Valenti, and Superti (2005), bLF prevented echoviruses-infected cells from apoptosis and inhibited viral replication. Echoviruses are small non-enveloped, single-stranded RNA viruses of the enterovirus type.

Mechanism of action

In conclusion, two antiviral mechanisms of LF have been proposed. First, LF appears to interact with the receptors on the cell surface, such as glycosaminoglycans (Mann, Romm, & Migliorini, 1994; Wu, Monroe, & Church, 1995), which are the binding sites for many viruses (Roderiquez et al., 1995; WuDunn & Spear 1989). Second, LF binds directly to viral particles and inhibits viral adsorption to target cells (Marchetti et al., 1996; Superti et al., 1997; Swart, Harmsen, et al., 1996; Yi et al., 1997). Glycosaminoglycans are long polyanionic carbohydrate chains that consist of repeating disaccharide units containing sulphate residues (Di Biase et al., 2003). Glycosaminoglycans are often covalently linked to a protein core to form proteoglycans, which are involved in several important functions, such as cell attachment, proliferation, migration, morphogenesis, and receptor-mediated endocytosis (Ho, Broze, & Schwartz, 1997; Poole, 1986). A number of viruses, including HIV, HSV, CMV and adenovirus, recognise cell-surface proteoglycans (heparin and heparan sulphate) as receptors (Di Biase et al., 2003; Rostand & Esko, 1997). Bovine LF has been shown to be more active than hLF against viruses; apo-LF is less effective than the iron saturated form (Valenti et al., 1998). The iron-saturated LF with a more compact conformation may have higher affinity for LF receptors on eukaryotic cells than the apoform (Davidson & Lonnerdal, 1989) and be more resistant to denaturation and to proteolysis (Harmsen et al., 1995). The differences in amino acid sequence of antiviral region, glycan chains and the number of disulphide bridges between hLF and bLF are likely to contribute to their differences in antiviral effectiveness (Valenti et al., 1998).

Abbreviations are: CHV, canine herpesvirus; FCV, feline calicivirus; FHV-1, feline herpesvirus type 1; GMP, glycomacropeptide (f106–169 of k-casein); HBV, hepatitis B virus; HCV, hepatitis C virus; HCMV, human cytomegalovirus; HIV-1, human immunodeficiency virus type 1; HPV, human papillomavirus; HSV-1 and HSV-2, herpes simplex virus type 1 and 2, respectively; Ig concentrate, immunoglobulin concentrate; LF, lactoferrin; bLF, bovine lactoferrin; hLF, human lactoferrin; Lfcin, lactoferricin; PV, poliovirus; RSV, respiratory syncytial virus.