Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses

Abstract

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Keywords: HSV-1; NMR; SARS-CoV-2; adenovirus; antiviral; magnesium chloride; modified heparin.

Figure 1

The effects of different concentrations of heparin, enoxaparin and MgCl₂/MgSO₄ on human adenovirus type 5 (HAdV5). Virus and further components were mixed pre-transduction and added to CHO-K1 cells. The luciferase measurements were performed 26 h post-transduction. All experiments included a positive control referring to untreated HAdV5: (a) CHO-K1 cells were infected with 1000 viral particles per cell with HAdV5 in the presence of different international units (I.U.) of heparin and enoxaparin; (b) Transduction rates of HAdV5 in the presence of increasing amounts of MgCl₂; (c) Infectivity of HAdV5 at 1000 viral particles per cell in the presence of different amounts of heparin and 5 µmol of MgCl₂; (d) Effects of MgCl₂ and MgSO₄ on HAdV5 transduction rates. Experiments were performed in triplicates and repeated three times. The mean standard errors are shown.

Figure 2

NMR analyses of magnesium chloride modified heparin: (a) Comparison of a portion of 1D ¹H NMR spectra of heparin (I), heparin–Mg²⁺ (II), and heparin–Ca²⁺ (III) acquired in 99.95% D₂O at 37 °C. Proton chemical shift assignment of iduronic acid, and glucosamine, residues are shown on top of each resonance. (IV) Bar plot showing ¹H chemical shift perturbation of heparin in the presence of Mg²⁺ and Ca²⁺. ¹H chemical shift difference between heparin–M²⁺ (M = Mg, Ca) and heparin are presented on the y-axis. Positive value indicates the deshielding of respective protons in the presence of M²⁺ (M = Mg, Ca). (V) Structure of the repeating disaccharide (iduronic acid-glucosamine) unit of heparin; (b) A portion of 2D NOESY spectra of heparin (I), heparin–Mg²⁺ (II), and heparin–Ca²⁺ (III) in 99.95% D₂O at 37 °C. Assignments are shown for the characteristic NOE cross-peaks. (IV) Bar plot representation of intra- and inter-residue NOE cross peak volumes. (V) Change in NOESY cross peak intensities of inter-residue A1-I4 and A1-I3 protons of heparin (blue), heparin–Mg²⁺ (cyan), and heparin–Ca²⁺ (orange).

Figure 3

Antiviral effect of modified heparin: (a) Modified heparin inhibits virus transduction of human adenovirus 5 (HAdV5) in HeLa cells. The mean fluorescent intensity (MFI) shows significantly lower transduction efficiencies after treatment with modified heparin compared to untreated control. For statistical analyses one-way ANOVA was performed, displayed are means + standard deviation. ** p-values ≤ 0.005; **** p-values ≤ 0.00005; (b) Measurements of herpes simplex virus-1 (HSV-1) on Vero cells showed decreased transduction efficiencies after treatment with heparin/MgCl₂ at different time points: t = −24 h (24 h before transduction), t = 0 h (at the time point of transduction) and t = +1 h (1 h after transduction). As control we compared the results with aciclovir treated cells (t = 0 h). Percentage of GFP-positive cells and mean fluorescent intensity (MFI) are displayed. For statistical analyses one-way ANOVA was performed, displayed are means + standard deviation. **** p-values ≤ 0.00005. pos. control: untreated virus; neg. control: cells without virus; RLU: relative light units; vpc: viral particle numbers per cell; (c) Antiviral effect against SARS-CoV-2. Viral load in vitro in Vero E6 cells 24 h post-transduction with SARS-CoV-2 in the presence or absence of magnesium-modified heparin. Vero E6 cells supplemented either with or without modified heparin were infected with 5 × 101 TCID50 of SARS-CoV2 per well (day 0); (d) Viral load in the cell culture supernatants analyzed by RT-qPCR (left panel) and RT-ddPCR (right panel) 24 h post-transduction. Supernatants of infected Vero E6 cells from experiment c, cultivated in the presence/absence of magnesium-modified heparin were collected and subjected to RT-qPCR and RT-ddPCR analysis. Experiments are from n = 10 biological replicates. Evaluation and graphical representation were carried out with GraphPad (Version 8.3.1, San Diego, CA, USA), p-values from student t-test (equal distribution, two-sided): **** p < 0.00005 and ** p < 0.005.