Targeting Host Cell Surface Nucleolin for RSV Therapy: Challenges and Opportunities

Nucleolin (NCL) has been reported as a cellular receptor for the human respiratory syncytial virus (RSV). We studied the effects of re-purposing AS1411, an anti-cancer compound that binds cell surface NCL, as a possible novel strategy for RSV therapy in vitro and in vivo. AS1411 was administered to RSV-infected cultures of non-polarized (HEp-2) and polarized (MDCK) epithelial cells and to virus-infected mice and cotton rats. Results of in vitro experiments showed that AS1411, used in micromolar concentrations, was associated with decreases in the number of virus-positive cells. Intranasal administration of AS1411 (50 mg/kg) to RSV-infected mice and cotton rats was associated with partial reductions in lung viral titers, decreased virus-associated airway inflammation, and decreased IL-4/IFN-γ ratios when compared to untreated, infected animals. In conclusion, our findings indicate that therapeutic use of AS1411 has modest effects on RSV replication and host response. While the results underscore the challenges of targeting cell surface NCL as a potential novel strategy for RSV therapy, they also highlight the potential of cell surface NCL as a therapeutic target.


Introduction
The respiratory syncytial virus (RSV, or human orthopneumovirus due to a recent name change [1]), an enveloped, single-stranded RNA Pneumovirus which affects all age groups worldwide and causes serious lower respiratory infections such as bronchiolitis and pneumonia [2]. Despite considerable efforts made since the 1960s to develop a safe and effective RSV vaccine, no licensed vaccine is available, and the recent failure of a phase III clinical trial of a vaccine candidate exemplifies the longstanding difficulties experienced in this area [3]. Palivizumab, an antibody that binds to the RSV fusion (F) protein (a viral envelope protein involved in initial viral entry into cells and cell-to-cell spread of infection), is used for RSV prophylaxis in selected "high-risk" patients, and there are reports of RSV resistance developing with respect to this drug [4][5][6][7]. Treatment of RSV infections has predominantly focused on targeting viral components such as the F protein [8] or the polymerase enzyme involved in viral nucleic acid replication [9]. Targeting viral components confers selection pressure for the emergence of drug-resistant viruses [5,10,11].
As obligate intracellular parasites, viruses interact with host cellular components during infection [12,13]. "Targeting the host" is a potential alternative strategy for anti-viral therapy, with this approach conferring a lower probability of the development of viral drug resistance [14]. With our discovery of nucleolin (NCL) as a functional cellular receptor for RSV that putatively interacts with F protein [15], we reasoned that administration of a compound that binds cell surface NCL might be useful for RSV treatment. To test this, we studied the effects of AS1411 (also known as ACT-GRO-777; ACT Corporation, Lexington, KY, USA), a synthetic DNA aptamer that binds cell surface NCL [16,17] and is in the process of phase II clinical trials for cancer treatment [18], on RSV infections in vitro and in vivo. In vitro experiments consisted of AS1411 administration to RSV-infected HEp-2 cells, a non-polarized cell line, or MDCK cells, a polarized cell line that expresses NCL on the apical aspect [19] and models the route of entry of RSV in the respiratory tract [15,[20][21][22][23]. In vivo experiments to investigate proof of concept involved RSV challenge, followed by AS1411 administration, in two rodent models: mice [24], and cotton rats [25]. The specific aims of this study were: (1) To determine the effects of AS1411 administration on RSV infection in HEp-2 or MDCK cells; and (2) To determine the effects of AS1411 given therapeutically to RSV-infected mice and cotton rats on levels of infectious virus in the lungs, animal body weight, virus-associated airway inflammation, host IL-4/IFN-γ ratio (an index of Th2 (IL-4), and Th1 (IFN-γ) used in evaluation of new vaccines and drugs [26][27][28]) and bronchoalveolar lavage (BAL) cytology. Figure 1 shows the schematic outline of the design for in vitro and in vivo experiments. In vitro experiments focused on quantifying HEp-2 or MDCK cells that showed RSV-associated positive immunofluorescence over a range of doses of AS1411. These conditions were chosen to model the in vivo situation in which the virus is not washed off and drug treatment would be given after the virus has already established infection and started spreading to neighboring cells. For in vivo studies, a single dose of AS1411 suspension (50 mg/kg) was delivered intranasally to anesthetized animals. This dose of AS1411 was based on dosing used in humans for cancer treatment, and the intranasal route reflected cell surface NCL being expressed on the apical aspect of airway epithelial cells [20,21].

In Vitro Experiments: Administration of AS1411 to Rsv-Infected HEp-2 Cells or MDCK Cells
HEp-2 cells or MDCK cells, cultured in EMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics, were plated onto black, flat, clear-bottomed 96 well plates (Thermo Fisher Scientific, Waltham, MA, USA) (1 × 10 4 cells/well). The next day RSV A2 (ATCC, Manassas, VA, USA), prepared by centrifugation as previously described [29], was added (multiplicity of infection (MOI)) = 0.1) and incubated for 24 h at 37 • C and 5% CO 2 to allow at least one full round of virus replication. Twenty-four hours later, AS1411 was applied to the wells in triplicate, starting at 70 µM with serial dilutions down to 4.4 µM. As a negative control we used a cytosine-rich derivative of the same compound (CRO) that does not interact with NCL.

Mouse Experiments
Animal studies were approved by the Hospital for Sick Children (Toronto, ON, Canada) Animal Care Committee in accordance with standards of the Canadian Council on Animal Care (Project Approval Number: 34753). Female Balb/c mice, aged 6 to 8 weeks, were purchased from Charles River Laboratories (St. Constant, QC, Canada), kept in a specific pathogen-free environment, and fed food and water ad libitum. The RSV used (RSV-A2, ATCC, Manassas, VA, USA) was propagated in HEp-2 cells and purified over sucrose in an ultracentrifuge [31].
For all virus instillation procedures, lightly sedated mice (isoflurane) readily inhaled 50 μL of instillate applied to their nares with a P-200 pipetter while the mouth was held closed. On day 0, mice received 5 × 10 6 plaque forming units of RSV A2 via intranasal installation (n = 4-6 per group). Mice were monitored and weighed daily.
On day 1 after RSV challenge, isoflurane-anaesthetized mice received AS1411 (50 mg/kg, diluted in PBS) via intranasal installation. The control group received buffer only. On day 4, when RSV titers were maximal in the mouse model [24], designated mice were euthanized and the lungs removed, weighed and homogenized for viral plaque assays on HEp-2 cell monolayers. On day 6, when RSVassociated airway inflammation is maximal in mice [24], designated animals were euthanized and underwent BAL for enumeration of inflammatory cells and measurement of protein levels of IL-4 and IFN-γ by a commercial assay (Luminex Molecular Diagnostics, Toronto, ON, Canada). Lungs were then removed, inflated and fixed in 10% neutral buffered formalin and processed for

Mouse Experiments
Animal studies were approved by the Hospital for Sick Children (Toronto, ON, Canada) Animal Care Committee in accordance with standards of the Canadian Council on Animal Care (Project Approval Number: 34753). Female Balb/c mice, aged 6 to 8 weeks, were purchased from Charles River Laboratories (St. Constant, QC, Canada), kept in a specific pathogen-free environment, and fed food and water ad libitum. The RSV used (RSV-A2, ATCC, Manassas, VA, USA) was propagated in HEp-2 cells and purified over sucrose in an ultracentrifuge [31].
For all virus instillation procedures, lightly sedated mice (isoflurane) readily inhaled 50 µL of instillate applied to their nares with a P-200 pipetter while the mouth was held closed. On day 0, mice received 5 × 10 6 plaque forming units of RSV A2 via intranasal installation (n = 4-6 per group). Mice were monitored and weighed daily.

Cotton Rat Experiments
Four-to-five-week-old cotton rats arrived one week after ordering, were quarantined for 14 days, and were randomized and assigned to treatment or control groups (n = 6 animals per group). Animals

Cotton Rat Experiments
Four-to-five-week-old cotton rats arrived one week after ordering, were quarantined for 14 days, and were randomized and assigned to treatment or control groups (n = 6 animals per group). Animals were maintained in accordance with an approved protocol from the local Institutional Review Board at the Lovelace Respiratory Research Institute (Albuquerque, NM, USA) (Project Approval Number: FY14-107).
On day 0, animals were lightly sedated with isoflurane (Millipore Sigma, Temecula, CA, USA) and received 100 µL containing either 1 × 10 6 pfu of RSV or uninfected sham inoculum intranasally. Animals were allowed to recover and then 4 h later, cotton rats were again anaesthetized with isoflurane, and received either AS1411 (50 mg/kg) or buffer, administered via intranasal installation. Animals were monitored and weighed daily. On day 5 post-RSV challenge, when lung viral titers in cotton rats are near peak levels and virus-associated lung inflammation can be discerned histologically [25], animals were euthanized and BAL was performed. Lung tissue was then removed and was either weighed and homogenized for viral titers, or snap frozen to isolate RNA, or inflated and fixed with 10% formalin and processed for preparation of paraffin-embedded blocks. For lung histopathological assessment, 4-µm-thick sections were put onto glass slides, stained with H&E or PAS, and coded. For each animal, an airway inflammation score was generated as described above for mice, adapted for cotton rats.

Statistical Analyses
Unless otherwise indicated, we expressed results as mean ± SEM and compared groups by either Mann-Whitney U test; Kruskal-Wallis test, or two-way ANOVA with Bonferroni procedure to account for multiple comparisons (PRISM software version 6.0, GraphPad Software Inc., La Jolla, CA, USA, http://www.graphpad.com). We considered a p value of ≤0.05 to be statistically significant.

In Vitro Experiments
For RSV-infected HEp-2 cells that were treated with AS1411 or control compound, and analyzed by using a fluorescent focus assay, the results ( Figure 3a HEp-2 (c) and MDCK (f) experiments in parallel to assess for cellular toxicity. No difference is seen between AS1411 and CRO in terms of cellular proliferation/health, indicating there was no significant cytotoxicity (2-way ANOVA, with no significant difference between each set of points using Bonferroni procedure).

Mouse Experiments
The results of mouse experiments are shown in Figure 4. Concerning viral culture results of lung homogenates (Figure 4a), intranasal administration of AS1411, given 1 day after RSV challenge, resulted in a modest (mean: 0.3 log), statistically significant decrease in lung viral titer on Day 4, in comparison to the sham treatment (p = 0.03).  6). A total of 17.5 µM of AS1411 is sufficient to obtain a 60% decrease in RSV fluorescence 24 h after it is added to HEp-2 cells (b) (** p = 0.0039 and * p = 0.01 using Bonferroni procedure; 2-way ANOVA, p < 0.0001). Quantities of 17.5 µM and 70 µM of AS1411 are sufficient to obtain a 87% and 97% decrease in RSV fluorescence in MDCK cells (e), respectively (** p = 0.003 and * p = 0.03 respectively using Bonferroni procedure; 2-way ANOVA, p < 0.0001). The MTS assay (Promega TM , Madison, WI, USA) was performed for both HEp-2 (c) and MDCK (f) experiments in parallel to assess for cellular toxicity. No difference is seen between AS1411 and CRO in terms of cellular proliferation/health, indicating there was no significant cytotoxicity (2-way ANOVA, with no significant difference between each set of points using Bonferroni procedure).

Mouse Experiments
The results of mouse experiments are shown in Figure 4. Concerning viral culture results of lung homogenates (Figure 4a), intranasal administration of AS1411, given 1 day after RSV challenge, resulted in a modest (mean: 0.3 log), statistically significant decrease in lung viral titer on Day 4, in comparison to the sham treatment (p = 0.03).
Concerning mouse body weights over the course of the experiment, Figure 4b shows that RSV infection was associated with significant weight loss in comparison to uninfected animals, and this was not affected by AS1411 treatment. In uninfected animals, AS1411 administration did not significantly affect animal body weight.
Histopathological scores of airway inflammation on Day 6 after viral challenge (Figure 4c) were significantly lower in the RSV-infected, AS1411-treated group as compared to the RSV-infected, untreated animals (p = 0.009). AS1411 administration to uninfected mice had no significant effects on airway inflammation compared to those animals given sham inoculum.
On Day 6 post-RSV challenge, in BAL fluid specimens (Figure 4d), there was a significant decrease in IL-4/IFN-γ protein levels in RSV-infected, AS1411-treated animals in comparison to the group of infected animals that received sham inoculum (p < 0.05). AS1411 administration to uninfected animals did not significantly affect IL-4/IFN-γ ratios in BAL fluid specimens. BAL cell counts were also determined and results are shown in Table 1. RSV challenge was associated with significant increases in BAL total cells, percentage of granulocytes and lymphocytes, and an accompanying significant decrease in percentage of macrophages (p < 0.002). These RSV-associated findings were observed irrespective of AS1411 treatment status. Moreover, administration of AS1411 had no significant effect on the total cell counts or percentages of granulocytes, lymphocytes, and macrophages in BAL fluid specimens.   Concerning mouse body weights over the course of the experiment, Figure 4b shows that RSV infection was associated with significant weight loss in comparison to uninfected animals, and this was not affected by AS1411 treatment. In uninfected animals, AS1411 administration did not significantly affect animal body weight.
Histopathological scores of airway inflammation on Day 6 after viral challenge (Figure 4c) were significantly lower in the RSV-infected, AS1411-treated group as compared to the RSV-infected, untreated animals (p = 0.009). AS1411 administration to uninfected mice had no significant effects on airway inflammation compared to those animals given sham inoculum.
On Day 6 post-RSV challenge, in BAL fluid specimens (Figure 4d), there was a significant decrease in IL-4/IFN-γ protein levels in RSV-infected, AS1411-treated animals in comparison to the group of infected animals that received sham inoculum (p < 0.05). AS1411 administration to uninfected animals did not significantly affect IL-4/IFN-γ ratios in BAL fluid specimens. BAL cell counts were also determined and results are shown in Table 1. RSV challenge was associated with significant increases in BAL total cells, percentage of granulocytes and lymphocytes, and an accompanying significant decrease in percentage of macrophages (p < 0.002). These RSV-associated findings were observed irrespective of AS1411 treatment status. Moreover, administration of AS1411 had no significant effect on the total cell counts or percentages of granulocytes, lymphocytes, and macrophages in BAL fluid specimens.

Cotton Rat Experiments
Results of cotton rat experiments are shown in Figure 5. Concerning viral cultures (Figure 5a), the group of cotton rats that received AS1411 treatment 4 h after viral challenge had a pronounced (mean: 1 log), statistically significant decrease in lung RSV titer on Day 5 in comparison to RSVinfected, sham-treated animals (p = 0.002).
Over the course of the experiment, all animals studied appeared healthy, irrespective of RSV exposure status (as expected with this animal model [25]) or AS1411 treatment status. Cotton rats showed no significant change in body weight in association with RSV infection, irrespective of AS1411 treatment status, over the course of the experiment (Figure 5b).
Histopathological scoring of cotton rat airways (Figure 5c) revealed two main findings: (1) in RSV-infected animals, AS1411 treatment was associated with a significantly decreased airway inflammation score on day 5, in comparison to virus-infected animals that did not receive the compound (p = 0.04); (2) administration of AS1411 to uninfected animals did not significantly affect airway inflammation scores, in comparison to the group receiving buffer only. RSV-infected, AS1411treated animals had a significantly lower IL-4/IFN-γ mRNA ratio than the "RSV only" group ( Figure  5d).
For BAL fluid specimens collected on Day 5, the results (Table 1) indicate no significant associations between RSV infection status or AS1411 treatment status on total cell counts, or the percentages of macrophages, lymphocytes or granulocytes. (c) AS1411 treatment decreased RSV-associated airway inflammation (*** p = 0.009; Kruskal-Wallis test with Bonferroni procedure) and did not affect airway pathology in uninfected animals (NS = not significant); (d) In RSV-infected animals, AS1411 treatment was associated with a significantly decreased IL-4/IFN-γ ratio (* p < 0.05; Mann-Whitney u test), considered to be a beneficial anti-RSV host response [36,37].

Cotton Rat Experiments
Results of cotton rat experiments are shown in Figure 5. Concerning viral cultures (Figure 5a), the group of cotton rats that received AS1411 treatment 4 h after viral challenge had a pronounced (mean: 1 log), statistically significant decrease in lung RSV titer on Day 5 in comparison to RSV-infected, sham-treated animals (p = 0.002).
Over the course of the experiment, all animals studied appeared healthy, irrespective of RSV exposure status (as expected with this animal model [25]) or AS1411 treatment status. Cotton rats showed no significant change in body weight in association with RSV infection, irrespective of AS1411 treatment status, over the course of the experiment (Figure 5b).
Histopathological scoring of cotton rat airways (Figure 5c) revealed two main findings: (1) in RSV-infected animals, AS1411 treatment was associated with a significantly decreased airway inflammation score on day 5, in comparison to virus-infected animals that did not receive the compound (p = 0.04); (2) administration of AS1411 to uninfected animals did not significantly affect airway inflammation scores, in comparison to the group receiving buffer only. RSV-infected, AS1411-treated animals had a significantly lower IL-4/IFN-γ mRNA ratio than the "RSV only" group ( Figure 5d).
For BAL fluid specimens collected on Day 5, the results (Table 1) indicate no significant associations between RSV infection status or AS1411 treatment status on total cell counts, or the percentages of macrophages, lymphocytes or granulocytes.

Discussion
The results of this study show that AS1411 treatment is effective at reducing levels of RSV observed in three different model systems: HEp-2 cell cultures, MDCK cell cultures, and replicating virus isolated from the lungs of mice and cotton rats. AS1411 administration to HEp-2/MDCK cell cultures was not associated with significant cytotoxicity over the dose range studied. The difference in response between the two cell lines is likely due to the amount of cell surface NCL available and its relative turnover: HEp-2 cells grow rapidly and are non-polarized, while MDCK cells grow more slowly and are polarized with cell surface NCL localized to the apical aspect [19]. In RSV-infected mice and cotton rats, AS1411 administration was associated with decreased airway inflammation and with decreased IL-4/IFN-γ ratios, both of which are considered to reflect a beneficial host response to virus infection. AS1411 administration to uninfected animals did not significantly affect animal body weight or BAL cytology in either species. Overall, these findings support the proof of principle of targeting cell surface NCL for the therapy of RSV infections.
In addition, our results provide further opportunity to extend previous work comparing the mouse and cotton rat models of experimental RSV lung infection [38]. In mouse experiments, AS1411 was given 1 day after inoculation with 5 × 10 6 pfu of RSV. This interval between virus challenge and

Discussion
The results of this study show that AS1411 treatment is effective at reducing levels of RSV observed in three different model systems: HEp-2 cell cultures, MDCK cell cultures, and replicating virus isolated from the lungs of mice and cotton rats. AS1411 administration to HEp-2/MDCK cell cultures was not associated with significant cytotoxicity over the dose range studied. The difference in response between the two cell lines is likely due to the amount of cell surface NCL available and its relative turnover: HEp-2 cells grow rapidly and are non-polarized, while MDCK cells grow more slowly and are polarized with cell surface NCL localized to the apical aspect [19]. In RSV-infected mice and cotton rats, AS1411 administration was associated with decreased airway inflammation and with decreased IL-4/IFN-γ ratios, both of which are considered to reflect a beneficial host response to virus infection. AS1411 administration to uninfected animals did not significantly affect animal body weight or BAL cytology in either species. Overall, these findings support the proof of principle of targeting cell surface NCL for the therapy of RSV infections.
In addition, our results provide further opportunity to extend previous work comparing the mouse and cotton rat models of experimental RSV lung infection [38]. In mouse experiments, AS1411 was given 1 day after inoculation with 5 × 10 6 pfu of RSV. This interval between virus challenge and drug treatment was associated with a modest (0.3 log) reduction in lung viral titer in AS1411 treated vs. untreated, RSV-infected groups on Day 4, when RSV replication is maximal in the mouse model. Cotton rats challenged with a lower viral inoculum (1 × 10 6 pfu) and given the same dose of AS1411 at an earlier time-point (4 h) than mice after RSV challenge, showed, as might be predicted, a more profound reduction lung viral titer (1 log) on Day 5. RSV-challenged mice lost approximately 20% of body weight over the course of the experiment, whereas all groups of cotton rats experienced similar degrees of weight gain. Concerning airway histopathology, both species showed significant RSV-associated increases in airway inflammation scores that were improved by AS1411 treatment (Figures 3c and 4c). Of note, baseline airway pathology scores for the uninfected control groups in the mice were near zero (Figure 3c), whereas cotton rats had some very mild baseline airway inflammation ( Figure 4c) and higher baseline BAL total cell concentrations (Table 1). Mice, but not cotton rats, showed significant RSV-associated increases in BAL total cell concentrations and changes in the percentages of infiltrating granulocytes, lymphocytes and macrophages (Table 1). These apparent species differences may reflect the use of inbred mice and outbred cotton rats, the latter of which are expected to have greater genetic variability in host inflammatory responses [38]. Irrespective of the differences observed between mice and cotton rats, the effect of AS1411 treatment was similar in both species. In particular, the extent of decrease of RSV-associated airway inflammation with AS1411 treatment (~30-40%) was similar in both species, when compared to virus-infected, untreated animals (Figures 3c and 4c). Further, AS1411-associated decreases in IL-4/IFN-γ ratios were similar in mice and cotton rats (Figures 3d and 4d), even though the cytokines were measured differently: in mice, BAL fluid specimens were tested for IL-4 and IFN-γ protein levels while in cotton rats, mRNA levels in the lungs were measured.
The modest effects observed of treating both mice and cotton rats with relatively large quantities of AS1411 may relate to the fact that the uptake of AS1411 into the cell is not just through the binding and internalization of cell surface NCL but also through other mechanisms [39]. In light of this, it may be advantageous to develop compounds, such as antibodies or other small molecules, that bind and block cell surface NCL primarily and prevent viral fusion. Also, studies in mice and rats with radiolabelled AS1411 show that over 63% AS1411 ends up excreted in the urine within hours of a single intravenous injection [40]. This rapid clearance of AS1411 has meant that, in order to achieve clinically relevant dosing for cancer therapy, regimens such as 7-day continuous intravenous infusions have been required [39]. In light of this last point it seems very likely that further optimization of the dosage regimen of this compound for RSV treatment will be useful.
The mouse and cotton rat experiments involved administration of single doses of AS1411 to establish proof of concept of this therapeutic strategy. Experiments designed to test different doses and dosing regimens of AS1411 can lead to a greater understanding of the effectiveness of targeting NCL in RSV therapy, providing characterization of safety profiles, and determining whether drug resistance can develop through this approach. For RSV, development of drug resistance is a relevant consideration, as instances of resistant viruses have been documented in association with the use of the RSV fusion inhibitor GS-5806 [41], and with palivizumab [4][5][6][7], both of which target the viral F protein.
Interestingly, there are an increasing number of reports indicating that NCL is involved in the replication of a variety of different viruses [42][43][44][45]. In particular, AS1411 has also been shown to inhibit HIV-1 attachment to the host cell [46]. In this work the investigators observed an approximately 60% inhibition of HIV infection using non-polarized cells, similar to our observations in HEp-2 cells, with AS1411 administration in the 0.4 µM to 50 µM range. As such, therapeutics developed around NCL targeting in the host may have broader applications beyond RSV.
Recently, there has been a major resurgence in the area of RSV vaccine development, and promising candidates continue to be developed [47], such that a successful vaccine may be imminent in the next few years. Even so, vaccines will not be useful in cases of acute infection, or in individuals for whom immunization may be contraindicated. Moreover, as has been established for other viruses such as HIV [48] and the hepatitis C virus [49], a multi-drug cocktail may well prove superior to monotherapy for RSV treatment [50]. In conclusion, our current work with AS1411 provides evidence that targeting host NCL is a novel, potentially viable strategy for treating RSV infections.