Lactoferrin inhibits enterovirus 71 infection by binding to VP1 protein and host cells

Abstract

The antiviral activities of bovine lactoferrin (LF) against enterovirus 71 (EV71) were studied both in vitro and in vivo. LF protected both human rhabdomyosarcoma and neuroblastoma SK-N-SH cell lines from EV71 infection when it was added at the same time, before, or within 30 min after EV71 infection. Using enzyme-linked immunosorbent assay-based binding assay and indirect fluorescent stain, we found that LF could bind to the target cells. Furthermore, it was found that LF could bind to the VP1 protein of EV71, which was blocked in the presence of anti-VP1 antibody. In addition, LF could induce IFN-α expression of SK-N-SH cells and inhibit EV71-induced IL-6 production. Finally, LF protected mice against lethal EV71 challenge. Taken together, these results suggest that LF can inhibit EV71 infection by interacting with both EV71 and host cells.

Introduction

Enterovirus 71 (EV71) is a positive-stranded RNA virus belonging to the enterovirus genus of the Picornaviridae family (Ho, 2000, McMinn, 2002). It is transmitted from person to person mainly by the fecal–oral route. After replication in the mucosal system, the virus may enter the circulation (viremia) and finally find its way to the central nervous system (Li et al., 2002). The clinical manifestations caused by EV71 infection vary from mild hand, foot and mouth disease or herpangina to aseptic meningitis, encephalitis, pulmonary edema and death. In 1998, an epidemic of EV71 infection affected more than 90,000 children in Taiwan and caused 78 deaths (Ho et al., 1999, Liu et al., 2000). There is still no vaccine or antiviral drug available against this infection. Recently, lactoferrin (LF) has been shown to be able to inhibit EV71 in vitro (Lin et al., 2002). However, the mechanism of LF to inhibit EV71 is still unclear.

LF, an iron binding glycoprotein with a molecular mass of ∼80 kDa, consists of 692 amino acids. LF of different species has high degree of sequence homology. LF is present on mucosal surfaces, within the specific granules of neutrophils, and in biological fluids including breast milk, saliva and seminal fluid, indicating that it may play a protective role in the innate immune response. Breast milk is by far the most abundant source of LF. Human colostrums contain up to 7 mg/ml of LF. The concentration of LF, however, varies greatly in other human body fluids. It is as high as 2 mg/ml in tears and is approximately 1 μg/ml in blood. However, its concentration can increase to 200 μg/ml in blood in inflammatory situation (Masson and Heremans, 1971).

Numerous biological functions have been ascribed to LF such as iron-transportation, and anti-microbial properties against bacteria, fungi, and several viruses (Farnaud and Evans, 2003, Florisa et al., 2003, van der Strate et al., 2001). LF also displays an antiviral activity against both DNA and RNA viruses including rotavirus, herpes virus and HIV (Harmsen et al., 1995, van der Strate et al., 2001). Most of these studies indicate that the antiviral effect of LF lies in the early phase of infection. LF prevents virus from entering the host cells, either by blocking cellular receptors, or by binding to the virus particles directly (van der Strate et al., 2001). In this study, we demonstrated that LF inhibited EV71 infection by binding to both EV71 and host cells.