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Lactoferrin inhibits HIV-1 replication in vitro and exhibits synergy when combined with zidovudine

Viani, Rolando M.a; Gutteberg, Tore J.d; Lathey, Janet L.a; Spector, Stephen


aDepartment of Pediatrics, bCenter for Molecular Genetics, cCenter for AIDS Research, University of California, San Diego, La Jolla, CA, USA; and dDepartment of Microbiology, University Hospital of Tromso, Tromso, Norway.

Sponsorship: This work was supported in part by Public Health Service grants AI-27563, AI-39004, and the UCSD Center for AIDS Research (AI-36214) from the National Institute of Allergy and Infectious Diseases.

Received: 11 January 1999; revised: 12 March 1999; accepted: 16 March 1999.

Lactoferrin, an 80kDa glycoprotein with well-known antibacterial properties, is present in mammalian milk and in most exocrine fluids such as saliva, bile, pancreatic fluid and tears [1]. The antiviral properties of human milk have also been demonstrated, and one study [2] suggested that lactoferrin is active against HIV-1 infection. Among 440 mother-infant pairs in Malawi, pregnant women with lactoferrin levels below 0.91g/ml had a 28.6% perinatal HIV-1 transmission rate, compared with a 12.1% rate in mothers with serum lactoferrin levels above 2.54μg/ml, demonstrating a significant correlation between low maternal serum lactoferrin levels and the risk of perinatal HIV-1 transmission [3].

Despite considerable progress in the interruption of mother-to-infant HIV-1 transmission using zidovudine intervention, as yet there are no approaches to prevent vertical transmission that can be widely used in developing countries. Because human lactoferrin and bovine lactoferrin have known broad-spectrum antiviral properties, their ability to inhibit HIV-1 replication was examined in vitro. We further evaluated the effect of the combination of either human or bovine lactoferrin with zidovudine in blocking the spread of HIV-1 infection in peripheral blood mononuclear cell (PBMC) cultures.

Three syncytium-inducing (SI) and three non-syncytium-inducing (NSI) clinical isolates from HIV-infected pregnant women were evaluated. In brief, a standardized inoculum of 4000 TCID50/ml was used to infect human or bovine lactoferrin pre-treated 4×106phytohaemagglutinin-stimulated PBMC in triplicate in a 96-well plate, as a modified drug susceptibility assay [4]. Human and bovine lactoferrin were prepared with tissue culture medium at concentrations ranging from 50 to 0.31μM. Zidovudine was used at concentrations between 0.25 and 0.001μM. A third plate with a combination of human or bovine lactoferrin and zidovudine at a molar ratio of 200:1 was prepared for combination studies. Zidovudine, human lactoferrin and bovine lactoferrin IC50 values were calculated by using the median-effect equation [5]. For a given HIV-1 inhibition (90 and 75%) a combination index (Cl) of <1, 1 and >1 indicate synergism, additive effects and antagonism, respectively [6].

Concentrations of lactoferrin from 0.31 to 50μM exhibited increasing dose-dependent inhibition of HIV-1 p24 antigen production for all six (three SI and three NSI) HIV-1 isolates. The mean human lactoferrin IC50 for the six isolates was 9.6μM (range 2.5-15.8μM), whereas the mean bovine lactoferrin IC50 was 2.4μM (range 0.24-6.3μM; P=0.004). The mean zidovudine IC50 was 0.02μM (range 0.01-0.07μM). A dose-dependent inhibition was observed for the combination of human lactoferrin and zidovudine, with a Cl90 and a Cl75 <1 for the six isolates tested, with a range of 0.15-0.96 and 0.17-0.74, respectively. Similar dose-dependent inhibition was observed for the combination of bovine lactoferrin and zidovudine, with Cl90 and Cl75 <1 for the six isolates tested, with a range of 0.15-0.67 and 0.19-0.57, respectively (see Table 1). These results indicate synergism against HIV-1 when zidovudine is combined with either human or bovine lactoferrin.

Table 1

Table 1

Human and bovine lactoferrin have recently been found to inhibit a single SI strain of HIV-1 in the MR-4 cell line [2]. It was postulated that the activity of lactoferrin is associated with its ability to bind to heparin-like molecules at the cell surface interfering with the virus-cell fusion and binding. In our study, pretreatment of PBMC with human and bovine lactoferrin demonstrated potent in-vitro anti-HIV-1 activity of SI and NSI HIV-1 clinical isolates. Bovine lactoferrin compared with human lactoferrin had a fourfold lower mean IC50 value, suggesting a more potent anti-HIV-1 effect.

Breastfeeding is not recommended in HIV-infected women in the industrialized world because of the risk of HIV-1 transmission; however, in developing countries breastfeeding is still common where there is no safe alternative. Milk lactoferrin levels are high in colostrum and then gradually decrease during lactation [7]. The decrease in lactoferrin correlates with the increasing risk of HIV-1 transmission through breastmilk as the infant grows older [8]. Lactoferrin supplement during this period may reduce the risk of HIV transmission.

Zidovudine given during pregnancy and to the neonate for 6 weeks has been shown to decrease HIV perinatal transmission by 68% [9]. Recently, a 50% decrease in perinatal HIV-1 transmission was demonstrated when zidovudine was administered during the last 4 weeks of pregnancy [10]. Suitable agents to be used in conjunction with zidovudine should have different targets, and be safe and inexpensive for use in developing countries. These findings suggest that human and bovine lactoferrin have anti-HIV-1 activity, when combined with zidovudine, demonstrate synergism against SI and NSI HIV-1 clinical isolates, and may prove useful in decreasing the perinatal transmission of HIV-1.

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