Development of Broad-Spectrum β-Cyclodextrins-Based Nanomaterials Against Influenza Viruses

Abstract

In recent decades, epidemics and pandemics have multiplied throughout the world, with viruses generally being the primary responsible agents. Among these, influenza viruses play a key role, as they potentially cause severe respiratory distress, representing a major threat to public health. Our study aims to develop new broad-spectrum antivirals against influenza to improve the response to viral disease outbreaks. We engineered macromolecules (named CD-SA) consisting of a β-cyclodextrin scaffold modified with hydrophobic linkers in the primary face, onto which unitary sialic acid epitopes are covalently grafted to mimic influenza virus-host receptors. We assessed the antiviral efficacy, mechanism of action, and the genetic barrier to resistance of this compound against influenza in vitro, ex vivo, and in vivo. We demonstrated that CD-SA, with a unitary SA, without extensive polysaccharides or specific connectivity, acts as a potent virucidal antiviral against several human influenza A and B viruses. Additionally, CD-SA displayed antiviral activity against SARS-CoV-2, a virus that also relies on sialic acid for attachment. We then assessed the genetic barrier to resistance for CD-SA. While resistance emerged after six passages with CD-SA alone, the virus remained sensitive through eight passages when co-treated with interferon-λ1 (IFN λ1). Finally, we completed the characterization of the antiviral activity by conducting both ex vivo and in vivo studies, demonstrating a potent antiviral effect in human airway epithelia and in a mouse model of infection, higher than that of Oseltamivir, a currently approved anti-influenza antiviral. The findings presented in this study support the potential therapeutic utility of a novel β-cyclodextrin-based nanomaterial for the treatment of influenza infections and potentially other sialic acid-dependent viruses.

Keywords: antiviral; broad‐spectrum; influenza; nanomaterials; virucidal; β‐cyclodextrins.

Figure 1

Schematic representation of CD‐SA and CD‐6'SLN. A β‐CD core is modified on its primary face with hydrophobic undecyl linkers exposing either a single sialic acid residue for CD‐SA (A) or 6'SLN (6’‐Sialyl‐N‐acetyllactosamine), a trisaccharide terminating with an α2‐6 sialic acid for CD‐6'SLN (B) [10].

Figure 2

Antiviral action of CD‐SA against N09. Dose–response assay of CD‐SA against N09 in virus pretreatment condition (A) and efficacy depending on time of administration (B). In (B), MDCK cells were either pretreated with CD‐SA for 1 h before infection; cotreated at the time of infection; or treated with CD‐SA 1 h after infection. Percentages of infection are shown as mean and SD. p‐values (*≤ 0.1; **≤ 0.01; ***≤ 0.001; ***≤ 0.0001) were calculated using two‐way ANOVA.

Figure 3

Virucidal activity of CD‐SA against N09. (A) Schematic representation of the protocol. (B) Virucidal assay: N09 and CD‐SA (EC₉₀ = 57 ng/mL) or the CD‐SA vehicle (UTR) were incubated for 3 h at 37°C and serially diluted onto MDCK cells and viral titers were evaluated by immunocytochemistry. A reduction of viral titer by > 1 log indicates a virucidal mechanism of action [10]. The ***p ≤ 0.001 was determined using an unpaired t‐test. (C) RNA exposure assay: Cycle threshold difference in N09 RT‐qPCR between RNase‐treated (SDS) virus molecule mixture (100 µg/mL) and buffer‐treated virus molecule mixture (untreated condition = UTR). The **p ≤ 0.01 was determined using an ordinary one‐way ANOVA. (D) Negative staining electron microscopy of N09 incubated with a control medium or 1.7 μg/mL of nonfunctionalized CD, or virucidal CD‐SA for 1 h at 37°C. Scale bar: 50 nm. (E) Envelope integrity assay: Relative fluorescence unit of R18‐labeled N09 measured after 3 h incubation with nonfunctionalized CD, triton X‐100, and CD‐SA. ****p ≤ 0.0001 were determined using one‐way ANOVA.

Figure 4

Antiviral efficacy of CD‐SA and CD‐6'SLN against N09 in ex vivo human airway epithelia (HAE) reconstituted at the air‐liquid interface. (A) Schematic of ex vivo HAE infection and treatment. (B) Tissues were infected apically and antivirals were administered from 1 to 4 dpi. Cell viability of uninfected treated controls was assessed by resazurin assay (dashed lines). Viral loads were daily quantified by RT‐qPCR. The results represent the mean and SD from two independent experiments. Statistics were obtained by analyzing the AUC (area under the curve). *p ≤ 0.1; **p ≤ 0.01.

Figure 5

Efficacy of CD‐SA and oseltamivir (OS) against influenza A/California/04/2009 in BALB/c mice. Female mice (10/group) were infected and intranasally treated with sterile saline (vehicle) or CD‐SA after 1 (A) or 2 dpi (B) and continued for 5 days. Control mice were treated by oral gavage with OS, 2 h before infection and twice daily afterwards for 5 days. Survival curves (left panels) and percentages of weight changes (right panels) are shown. Survival rates were compared by the Mantel−Cox log‐rank test using Prism 9 (GraphPad Software, San Diego, CA). ***p < 0.001, *p < 0.05 compared to the vehicle‐treated animals. The mean weight of surviving mice at the different days postinfection was computed and plotted.