Viral infections kill millions of people and new antivirals are needed. Nontoxic drugs that irreversibly inhibit viruses (virucidal) are postulated to be ideal. Unfortunately, all virucidal molecules described to date are cytotoxic. We recently developed nontoxic, broad-spectrum virucidal gold nanoparticles. Here, we develop further the concept and describe cyclodextrins, modified with mercaptoundecane sulfonic acids, to mimic heparan sulfates and to provide the key nontoxic virucidal action. We show that the resulting macromolecules are broad-spectrum, biocompatible, and virucidal at micromolar concentrations in vitro against many viruses [including herpes simplex virus (HSV), respiratory syncytial virus (RSV), dengue virus, and Zika virus]. They are effective ex vivo against both laboratory and clinical strains of RSV and HSV-2 in respiratory and vaginal tissue culture models, respectively. Additionally, they are effective when administrated in mice before intravaginal HSV-2 inoculation. Lastly, they pass a mutation resistance test that the currently available anti-HSV drug (acyclovir) fails.

Cyclodextrins (CDs) are naturally occurring glucose derivatives, with a rigid cyclic structure, consisting of α(1-4)–linked glucopyranoside units. The most common CDs, referred to as α, β, and γ, have 6-, 7-, and 8-glucopyranoside units, respectively. They have found use in many commercial applications including drug delivery, air fresheners, cosmetics, and food ( 12 , 13 ). Sulfonated CDs have shown antiviral properties only against HIV ( 14 – 18 ); however, their action was found to be reversible (virustatic) and virus specific. Here, we attach highly sulfonated chemicals to a U.S. Food and Drug Administration (FDA)–approved CD scaffold and achieve highly efficient virucidal broad-spectrum molecules, effective in vitro, ex vivo, and in an animal model.

Recently, we have shown that highly sulfonated gold nanoparticles display broad-spectrum virucidal properties in vitro, ex vivo, and in vivo ( 10 ). In that work, a slight modification to the nanoparticle structure, relative to published work ( 6 ), altered the antiviral mode of action from virustatic to virucidal, with nanomolar median effective concentrations (EC 50 ’s). This allowed us to maintain the broad-spectrum and nontoxic properties of virustatic nanoparticles while imparting a virucidal mechanism. Unfortunately, there are concerns with the use of gold nanoparticles as drugs due to their unknown clearance mechanism and possible long-term toxicity ( 11 ). Nonetheless, the principle of displaying multiple viral attachment ligands to create a virucidal drug remains compelling.

Broad-spectrum antivirals like heparin or heparin-like materials ( 5 – 8 ) have been developed that mimic the cell surface sugars responsible for initial viral attachment, such as heparan sulfate (HS). Unfortunately, upon dilution, the viruses are no longer bound to the drug and are still infectious; thus, the clinical efficacy remains unproven ( 2 – 4 ). A drug with an irreversible action, i.e., a virucidal drug, could be ideal to fight viral infection, given that it would not be subject to loss of efficacy upon dilution and have long-lasting effects. All previously identified virucidal molecules have toxic side effects that render their clinical use impossible ( 9 ). To the best of our knowledge, there is currently no approved drug that shows virucidal activity.

Viruses can negatively affect society at several levels: from viral infections of food crops and livestock to the serious health impacts of viruses that infect humans, such as HIV, Ebola, and Zika virus (ZIKV). When prevention is not possible, drugs must be administered to limit viral replication and aid the immune systems fight against the infection, if they are available. Unfortunately, most existing antivirals act intracellularly, with related problems of permeability and toxicity, are virus specific ( 1 ), and/or have a reversible (virustatic) effect ( 2 – 4 ).

RESULTS AND DISCUSSION

To test whether a modified CD (CD1; Fig. 1A) has antiviral activity like its nanogold counterpart (10), we used sodium undec-10-enesulfonate to synthesize a modified CD (CD1) (see figs. S1 and S2 for characterization) that exposes the sulfonate groups in a similar manner. In addition, two other modified CDs were synthesized. To alter the length of the linker, we synthesized CD2 (Fig. 1A), which bears a seven-carbon sulfonated alkyl chain (see figs. S1 and S3 for characterization). While CD1 showed strong inhibition of the growth of herpes simplex virus type 2 (HSV-2), with an EC 50 of 28.51 ± 2.319 μg/ml, CD2 showed no significant effect (Fig. 1B). To alter the nature of the linker, we also synthesized a nonalkyl linker of similar overall length to sodium undec-10-enesulfonate (CD3) (see figs. S1 and S4 for characterization). We hypothesized that removing the flexibility of the linker would result in stronger binding to the virus and improved overall antiviral effect.

Fig. 1 Structures and virucidal data for modified CDs. (A) Structures of modified CDs and relative effective concentrations of inhibition of HSV-2 growth. (B) Dose-response assay in Vero cells. Serial dilutions of CD1, CD2, and CD3 were incubated for 1 hour at 37°C with HSV-2 and then added on cells for 2 hours at 37°C. Subsequently, cells were washed and overlaid with medium containing methylcellulose. Plaques were counted 24 hpi. Percentages of infections were calculated by comparing the number of plaques in treated and untreated wells. (C) Virucidal assays: HSV-2 was incubated with media or CDs (CD1, CD2, and CD3) at 300 μg/ml and then serially diluted on Vero cells to a negligible concentration of compound. Results are shown as the mean and SEM of three (for CD1) and two (for all other compounds) independent experiments. UT, untreated; n.a., not assessable.

In a dose response assay, CD3 displayed enhanced antiviral effects, with an approximately threefold reduction in EC 50 over the time course of a single-cycle infection compared to CD1. To determine whether the observed inhibition of HSV-2 growth was virustatic or virucidal, we pretreated viral solutions with CD1 to CD3. CD1 was highly virucidal, but this effect was markedly reduced when moving from CD1 to CD3 (Fig. 1C). These findings for the first time assert the utility of long flexible linkers to provide a virucidal mode of action.

CDs are known to extract cholesterol from membranes (19). To exclude a cholesterol-dependent antiviral effect of CD1 related to this property, we performed a cholesterol replenishment assay in cells infected with HSV-2 and treated with CD1 or methyl-β-CD at their EC 90 . As shown in fig. S5, the inhibitory activity of methyl-β-CD, but not of CD1, is lost in the presence of cholesterol, highlighting that CD1 is antiviral in a cholesterol-independent manner.

To establish whether our functionalized CDs exhibited a broad-spectrum of antiviral activity comparable to the gold nanoparticles (10), we investigated their inhibitory effect against several HS-dependent viruses. As shown in Table 1, CD1 displayed broad-spectrum activity against a wide range of viruses belonging to different families. The molecule is active against HSV-1 as well as laboratory-passaged strains, clinical strains (passaged only once in cells), and acyclovir-resistant HSV-2. Antiviral activity is maintained against members of the Pneumoviridae family [respiratory syncytial virus type A (RSV-A) and B (RSV-B) and human metapneumovirus (HMPV)], against members of the Paramyxoviridae family [human parainfluenzavirus 3 (HPIV3)], and against members of the Flaviviridae family [dengue virus type 2 (DENV-2), ZIKV, and hepatitis C virus (HCV)]. For the latter, both the wild-type strain and variants resistant to protease inhibitors and NS5A inhibitors like BILN-2061 and daclatasvir (HCV D168A and HCV Y93H) (20) are susceptible to CD1. In addition, the compound proved to be active also in a fusion assay between cells expressing CD4 and cells expressing gp120-gp41 of HIV (21), demonstrating an ability to prevent this viral protein–receptor interaction. Enterovirus D68 (Fermon strain) and influenza virus H3N2, two viruses that were previously shown to be dependent on sialic acid, were not inhibited, supporting the specificity of the molecule (22). In all the different cell lines tested, no toxicity of the drug was evidenced up to 300 μg/ml, which corresponds to favorable selectivity indices (SIs). In contrast, Captisol, an FDA-approved sulfonated CD solubilizing reagent, displayed no antiviral activities.

Table 1 Inhibitory activity of CD1 on different viruses. Results are mean of two independent experiments. n.a., not assessable. View this table:

After having demonstrated its broad-spectrum antiviral activity, we evaluated the mechanism of action of CD1 through a time of addition assay using three different viruses (RSV-A, DENV-2, and HSV-2) (Fig. 2A). No activity was observed when cells were preincubated with compound and then washed out before infection, ruling out an indirect cell-mediated effect. Next, we evaluated the direct effect of the drug on the virus when added before, during, or after infection. The strongest inhibitory effect was observed when the compound was preincubated with the virus for 1 hour before infection, as shown in Fig. 2A. However, CD1 was active also when added directly during infection without prior incubation or after infection, opening possibilities for a therapeutic use (see also fig. S6). We subsequently verified, through virucidal assays, that the inhibition is linked to a permanent effect, which is not lost upon dilution (Fig. 2B). A DNA exposure assay further confirmed that after incubation with CD1, the HSV-2 genome becomes accessible to degradation by deoxyribonucleases (DNases), while nontreated viruses remain protected (Fig. 2C). We also observed a virucidal action against ZIKV (Table 1 and fig. S7), whose dependence on heparan sulfate proteoglycans (HSPGs) is debated (23–25).

Fig. 2 Mechanism of action of CD1. (A and B) CD1 was tested against RSV-A (top), DENV-2 (middle), and HSV-2 (bottom). (A) CD1 was either preincubated for 1 hour with the virus before addition on cells (Pretreatment virus), directly added with viruses on cells (During infection), added for 1 hour on cells and then washed out before infection (Pretreatment cells), or finally added after removal of the virus (Post-infection). (B) Virucidal assay: Viruses (in panels as above) were incubated with CD1 (30 μg) for 1 hour and then serially diluted on cells. (C) DNA exposure assay: HSV-2 was incubated in the presence or absence of CD1 (30 μg) for 1 hour at 37°C and then incubated for 30 min with Turbo DNase or only buffer and subsequently subjected to qPCR. Results are expressed in fold change of DNase treated versus untreated. (D) Time course of virucidal activity: HSV-2 and CD1 (30 μg) were incubated for different time periods and then serially diluted on Vero cells. (E and F) Drug resistance assay: HSV-2 passaged eight times in the presence of increasing concentrations of (E) acyclovir or (F) CD1, or no inhibitory compounds were subjected to a dose-response assay. The percentages of infection were calculated by comparing the number of plaques in treated and untreated wells. Results are mean and SEM of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005.

For HSV-2, we analyzed in depth the time dependency of the virucidal activity (Fig. 2D). It was possible to observe a significant reduction of viral titer after 5 min and a complete inactivation at 15 min. These results allow us to infer that because the time required to exert activity is short, CD1 is virucidal also when added after infection on the viral progeny released from infected cells and could inhibit cell-to-cell spread.

To further investigate the in vitro activity of CD1, its interaction with serum (26) was studied by comparing the EC 50 in its presence or absence. As shown in fig. S8, CD1 is inhibited by fetal bovine serum (FBS), which is demonstrated by the significant shift in the EC 50 , both against HSV-2 and RSV-A. In the absence of serum, CD1 was shown to have nanomolar activity against HSV-2 and low micromolar activity against RSV-A. These compounds are thus optimal for topical administration, while they need further improvement to treat systemic infections.

To investigate the structural factors likely to lead to virucidal effects of this broad-spectrum antiviral, we performed atomistic molecular dynamics (MD) simulations of the three modified CDs (Fig. 3A) interacting with glycoprotein B (gB), which is located on the surface of HSV-2 (see the Supplementary Materials). The structural rationale for these comparisons was that relatively short seven-carbon sulfonated alkyl chain reduced the number of interactions between the CD and virus, leading to its observed antiviral affect. We also hypothesized that the nature of the linker plays an important role in antiviral properties, exemplified by the antiviral activity of CD3, which bears a nonalkyl linker of similar overall length to sodium undec-10-enesulfonate in CD1. Note that this linker leads to a better reversible viral inhibition but that the inhibitory effect is almost totally lost after dilution (i.e., limits to no virucidal effect). This leads us to propose that the nature of the linker, independent of the binding affinity, is key to provide a virucidal mode of action.

Fig. 3 MD simulations. Interactions of CDs with gB proteins. (A) Representative snapshots of the modified CDs interacting with HSV-2 gB proteins after 50 ns of MD simulations. (B) Average distance between gB fusion loops, defined as the instantaneous average of the three distances labeled in fig. S9, over time. (C) Number of CDs interacting with gB proteins over time.

Figure 3 shows the results of CD1, CD2, and CD3 interacting with the gB protein, which were simulated by placing 10 CDs near the gB fusion loops (solvated in 0.15 M NaCl solution) before being released. Figure 3 (B and C) shows the number of binding CDs per gB and the distance between the gB fusion loops, respectively, after 50 ns. On average, CD1 bound to the most gB molecules over time, which induced a greater distance between gB fusion loops. Both CD2 and CD3 also interact with the fusion loops of gB, but they only cause small fluctuations in the average distance between the fusion loops. We propose that virucidal action of CDs relies on its ability to block the fusion loop, which subsequently leads to the induced gB conformational change (here, manifested by opening of the gB trimer). The stronger binding strength of CDs to gB does not guarantee larger binding numbers of CDs (fig. S9), because the interaction between CDs may cause the accumulation of CDs on the binding site.

The emergence of pathogen resistance to antimicrobial drugs is an important issue worldwide. One solution to drug resistance is the use of combined therapies, as is currently used in the treatment of HIV and HCV. Therefore, the identification of drugs with extremely high barriers to resistance is important. We compared the barrier for resistance to CD1 with that of acyclovir (an inhibitor of viral DNA polymerase, which is in clinical use) by culturing HSV-2 in the presence of increasing concentrations of the two molecules, using a procedure described previously (27). After eight passages in increasing concentrations of acyclovir (from 0.14 to 17.9 μg/ml), the EC 50 value was 86.9 times higher than that of a virus passaged in the absence of the drug, when tested for its acyclovir inhibition activity, compared to a virus passaged in the absence of acyclovir (Fig. 2E). In contrast, no infectious virus was recovered in supernatants of cells treated with CD1 (160 μg/ml) by passage 5. For passages 5 to 8, the concentration of CD1 was maintained at 80 μg/ml, and still poor viral growth was observed. Therefore, as opposed to acyclovir, the EC 50 value of CD1 did not change after eight passages in the presence of the drug, suggesting that under these experimental conditions, HSV-2 is unable to develop resistance to CD1.

As we have observed broad-spectrum virucidal activity and a high barrier to resistance in cultured cell lines, we tested whether the antiviral activity was maintained in three-dimensional (3D) tissue cultures derived from human biopsies and redifferentiated in vitro to mimic the 3D structure of human tissues. We evaluated the antiviral activity against RSV in an upper respiratory tissue model that reproduces the human upper airway epithelium with its characteristic mucus-secreting cells, ciliated cells, and basal cells (MucilAir, Epithelix). CD1 presented an efficiency comparable to palivizumab (an FDA-approved monoclonal antibody directed against RSV surface protein F) not only in co-treatment assays, where the drug and the virus were added simultaneously on the apical surfaces of tissues but also in therapeutic conditions where CD1 was added 24 hours post-infection (hpi), demonstrating the ability of CD1 to control the infection (Fig. 4, A and B). The complete protection in co-treatment assays was also confirmed by the absence of infected cells in treated tissues (Fig. 4D). CD1 caused neither toxicity nor release of proinflammatory cytokines even at high doses, or after repeated administrations, in this polarized respiratory tissue model (fig. S10). This tissue culture system also allows growth of viruses directly from infected specimens, thus avoiding potential artefacts that result from excessive adaptation to cell culture. We thus tested the ability of the compound to inhibit a clinical RSV-A isolate amplified only in reconstituted airway epithelia. Figure 4C shows that the inhibition is maintained also in strains currently circulating in the population. Of note, the inhibitory activity was reduced on clinical isolates compared to laboratory-adapted RSV isolates but remained comparable to that of palivizumab (Fig. 4C).

Fig. 4 Ex vivo experiments. (A) Co-treatment of respiratory tissues with RSV-A and CD1 (10 μg). (B) Respiratory tissues infected with RSV-A and treated with CD1 (6 μg) 24 hpi (Post-treatment). (C) Co-treatment of respiratory tissues with a clinical strain of RSV-A and CD1 (10 μg). (D) Respiratory tissues infected with RSV-A or cotreated with RSV-A and CD1 were subjected to immunostaining at 7 days post-infection (dpi). Green, ciliated cells (tubulin); red, infected cells; blue, nuclei. Scale bars, 20 μm. (E) Co-treatment of vaginal tissues with HSV-2 and CD1 (50 μg). (F) Vaginal tissues infected with HSV-2 and treated with CD1 (30 μg) 8 hpi post-treatment. (G) Co-treatment of vaginal tissues with a clinical strain of HSV-2 and CD1 (50 μg). Results are mean and SEM of two independent experiments. (H) Vaginal tissues infected with HSV-2 or cotreated with HSV-2 and CD1 were subjected to immunostaining at 7 dpi. Red, infected cells; blue, nuclei. Scale bars, 100 μm.

An additional possible application of these modified CDs, in line with their broad-spectrum antiviral activities, is as a vaginal microbicide to prevent and treat sexually transmitted infections such as HIV, HPV (human papillomavirus), and HSV-2. Therefore, we tested the ability of CD1 to inhibit HSV-2 in ex vivo vaginal tissues. We observed a good inhibition (Fig. 4, E and F) both in co-treatment, confirmed also by immunofluorescence (Fig. 4H) and in post-treatment; in the latter, CD1 was formulated in 2.7% hydroxyethylcellulose (HEC) gel and added 8 hpi. Moreover, the compound was also able to inhibit a clinical HSV-2 strain isolated from a vaginal swab and passaged only once in cells (Fig. 4G). Because ZIKV is sexually transmissible, we also evaluated the ability to inhibit this virus in this ex vivo model. The results presented in fig. S7 show that, in the presence of CD1, the replication of the virus is abrogated, demonstrating the broad-spectrum activity also ex vivo. These data show that the activity evidenced in vitro is translated in ex vivo human models. Last, we evaluated the efficacy of CD1 formulated in HEC gel to prevent intravaginal HSV-2 infection in a murine model. BALB/c mice pretreated with the CD1 formulation exhibited only a slight weight loss and a lower increase in mean lesion scores after HSV-2 infection compared to mice that received the HEC gel (Fig. 5, A and B). The area under the curve (AUC) for mean lesion scores calculated from days 4 to 10 after infection was lower in the CD1 compared to the HEC group (39.70 ± 25.14 and 54.05 ± 17.78, respectively). No toxicity (such as weight loss, redness, and swelling in the vaginal region) was recorded following one intravaginal application of 15 μl of the HEC gel or CD1 formulation to mice. The percent survival was higher in mice that received the CD1 formulation (30%) compared to the HEC group (10%), but the difference was not statistically significant (Fig. 5C). Viral titers in vaginal mucosa of mice pretreated with the CD1 formulation were significantly lower compared to mice that received the HEC gel (Fig. 5D). These data show that the administration of CD1 formulation before intravaginal challenge with HSV-2 delayed the infection of mice. This suggests that CD1 could have an interesting potential as a vaginal microbicide.