Antiviral Research 64 (2004) 63–68 Lactoferrin inhibits human papillomavirus binding and uptake in vitro Peter Drobni ∗ , Jonas N¨ aslund, Magnus Evander Department of Virology, Ume˚ a University, SE-901 85 Ume˚ a, Sweden Received 31 December 2003; accepted 24 May 2004 Abstract Lactoferrin (LF), a member of the transferrin family, is a bi-globular protein secreted in milk, saliva, tears, seminal fluid, endocervix and vaginal secretions. LF is an important player in the defence against pathogenic microorganisms and has also been shown to have activity against several viruses including herpesvirus, adenovirus, rotavirus and poliovirus. The antiviral activity of LF is directed against the early steps of viral infection and the LF antiviral effect against herpesvirus is mediated through LF binding to the herpesvirus receptor heparan sulfate. Human papillomavirus (HPV) causes genital warts and is a prerequisite for cervical cancer. HPV can also use heparan sulfate on the cell surface as a receptor. We studied the inhibition by LF on HPV entry by incubating HaCaT cells and HPV-16 virus-like particles (VLPs) with either human (HLF) or bovine lactoferrin (BLF). LF inhibited internalization of HPV-16 particles using CFDA-SE-labelled VLPs that only fluoresce after internalisation. By using a western blot assay we also found dose-dependent LF inhibition of HPV-16 VLP binding to the HaCaT cell surface. BLF was a more potent inhibitor of HPV entry than human LF. It was also clear that LF acted early in the HPV uptake process. © 2004 Elsevier B.V. All rights reserved. Keywords: Lactoferrin; Human papillomavirus; Heparan sulfate; Antiviral activity 1. Introduction Lactoferrin (LF) is a bi-globular (N- and C-lobe) protein consisting of a single polypeptide chain divided by a hinge region. The two globes can both bind one metal ion, such as Fe 2+ , Fe 3+ , Cu 2+ or Zn 2+ . The molecular weight of LF is 80 kDa and the gene shows a high degree of homol- ogy between different species. LF is a member of the trans- ferrin family and can be found in mucus secretions, milk, saliva, tears, seminal fluids and vaginal secretions. The high- est concentrations of LF is found in colostrum with concen- trations up to 7 g/l (Cohen et al., 1987). LF is expressed in human endocervix and by most of the endocervical glands (Farley et al., 1997). In human vaginal mucus the concentra- tion varies from 62.9 to 218 g/mg of protein after menses and 3.8 to 11.4 g/mg of protein before menses. Women tak- ing oral contraceptives show no variation in LF level during the menses cycle. This suggests hormonal control of LF lev- ∗ Corresponding author. Tel.: +46 90 7852879; fax: +46 90 129905. E-mail address: Peter.drobni@climi.umu.se (P. Drobni). els (Cohen et al., 1987). Furthermore, LF can be found in the secondary vesicles of neutrophilic granulocytes (Hansen et al., 1975). LF plays an important role in the primary defence against pathogenic microorganisms. It has antimicrobial activities against both Gram-negative and Gram-positive bacteria and fungicidal activities against Candida (Bellamy et al., 1993). It also has antiviral activities against rotavirus, herpesviruses, poliovirus, respiratory syncytial virus, hepatitis C virus, hu- man immunodeficiency virus (van der Strate et al., 2001), adenovirus (Di Biase et al., 2003), enterovirus 71 (Lin et al., 2002)), hepatitis B virus (Hara et al., 2002) and hantavirus (Murphy et al., 2001). Furthermore, lactoferricin, the trypti- cally cleaved N-terminal part of LF, has been shown to have bactericidal (Vorland et al., 1999), fungicidal (Bellamy et al., 1993) as well as antiviral activity. For virus inhibition lacto- ferricin is active against herpes simplex virus (Andersen et al., 2003), human cytomegalovirus (Andersen et al., 2001), aden- ovirus (Di Biase et al., 2003) and feline calicivirus (McCann et al., 2003). The interactions and capacities of LF to re- duce infections are exerted in different ways between virus 0166-3542/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2004.05.005