Effects of pH alteration on respiratory syncytial virus in human airway epithelial cells

Background Respiratory syncytial virus (RSV) is a leading cause of respiratory distress and hospitalisation in the paediatric population. Low airway surface pH impairs antimicrobial host defence and worsens airway inflammation. Inhaled Optate safely raises airway surface pH in humans and raises intracellular pH in primary human airway epithelial cells (HAECs) in vitro. We aimed to determine whether raising intracellular pH with Optate would decrease infection and replication of RSV in primary HAECs. Methods We cultured HAECs from healthy subjects in both air–liquid interface and submerged conditions. We infected HAECs with green fluorescent protein-labelled RSV (GFP-RSV; multiplicity of infection=1) and treated them with Optate or PBS control. We collected supernatant after a 4-h incubation and then every 24 h. We used fluorescence intensity, fluorescent particle counts, plaque assays, Western blots and ELISA to quantitate infection. Results In submerged culture, fluorescence intensity decreased in Optate-treated cells (48 h p=0.0174, 72 h p≤0.001). Similarly, Optate treatment resulted in decreased fluorescent particle count (48 h p=0.0178, 72 h p=0.0019) and plaque-forming units (48 h p=0.0011, 72 h p=0.0148) from cell culture supernatant. In differentiated HAECs cultured at ALI, Optate treatment decreased fluorescence intensity (p≤0.01), GFP via Western blot and ELISA (p<0.0001), and RSV-fusion protein via ELISA (p=0.001). Additionally, RSV infection decreased as Optate concentration increased in a dose-dependent manner (p<0.001). Conclusions Optate inhibits RSV infection in primary HAECs in a dose-dependent manner. These findings suggest that Optate may have potential as an inhaled therapeutic for patients with RSV.


Introduction
Respiratory syncytial virus (RSV) is a member of the Pneumoviridae family that causes airway damage and is the leading cause of severe lower respiratory tract infections in children [1,2]. In the United States, RSV accounts for >2 million outpatient visits and nearly 60 000 hospitalisations in children aged <5 years annually [3,4]. Currently, no effective therapies or vaccines exist for RSV, and while preventive antibodies are on the market, they remain restricted to a small group of high-risk infants [5]. A safe treatment for RSV would be beneficial throughout the world. pH serves as a trigger for penetration [7,8]. Additionally, many viruses require acidic endosomal pH for viral surface protein activation [9]. While RSV was thought to enter the cell in a pH-independent manner via direct membrane fusion and release of RSV nucleocapsids into the cytoplasm, recent studies have shown that clathrin function, macropinocytosis and endocytosis play a key role in the virulence of RSV [10,11]. Specifically, fusion protein cleavage via an endosomal protease that may require low pH for activation makes the virus infectious [11][12][13].
Optate (IND #139144) is a safe, glycine-based, inhaled buffer that alkalinises airway extracellular, intracellular and endosomal pH. We have demonstrated that exposing human airway epithelial cells (HAECs) to Optate also alters endosomal trafficking and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in primary HAECs [14,15]. Given the dependence of RSV on proteolytic cleavage of the fusion protein in the acidic endosome, we hypothesise that by increasing intracellular pH with Optate we will similarly inhibit RSV infection and replication.

Study design
The main objective of this study was to determine whether raising intracellular pH with Optate would decrease infection and replication of RSV in primary HAECs. Primary HAECs from three healthy, nonsmoking donors were grown as described previously under submerged conditions and at air-liquid interface (ALI) at passages 3 or 4 [16][17][18]. After optimising techniques with one donor under submerged conditions and at ALI, two additional donors were used for all other ALI experiments. Graphs with data from multiple donors are colour coded to allow for easy identification of each donor. Biological replicates are included in the datasets. HAECs were infected with RSV with green fluorescent protein (RSV-GFP; ViraTree, Research Triangle Park, NC, USA; product #R121, RSV-A2) at a multiplicity of infection of 1. Optate (at concentrations used in humans in vivo [14,15]) or control (PBS, pH 7.2) were co-administered with RSV-GFP to HAECs. Negative control groups were cells that were not infected with RSV and were not treated with Optate or PBS. Supernatant was harvested and stored at −80°C after a 4-h incubation period and then every 24 h for 3 days for submerged cells and up to 10 days as needed to achieve optimal infection for cells cultured at ALI. Submerged cells were treated with Optate or PBS daily with media change, while ALI cells were treated apically for 20 min twice daily.

Quantifying viral infection by fluorescence intensity
Fluorescence intensity quantification was calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/) on microscopic images obtained with the EVOS M5000 microscope (Thermo Fisher Scientific, Carlsbad, CA, USA) as described previously [14]. Images were taken while microscopy was focused on the most apical layer of epithelial cells every 24 h for either 3 days (submerged cells) or up to 10 days (cells cultured at ALI) using the Invitrogen GFP Light Cube (Thermo Fisher Scientific) prior to daily treatment with PBS or Optate. Fluorescence images were uploaded to ImageJ, image fields were selected, images were processed by subtracting the background using default settings, fluorescence intensity was analysed and average fluorescence intensity was compared between all groups. This technique reflects the level of RSV-GFP infection by quantifying fluorescence from the GFP in infected cells [19].
Fluorescent particle counting and plaque assays Vero E6 (African green monkey kidney cell line; ATCC, Manassas, VA, USA) were plated on the day prior to the experiment. Plaque assays were performed as described previously using the stored supernatant from the previous experiments outlined earlier [14]. Fluorescent particles were counted using fluorescence microscopy with Invitrogen GFP Light Cube on day 5 and results are reported as focus-forming units per millilitre.  Optate inhibits respiratory syncytial virus (RSV) infection and alters intracellular pH in a dose-dependent manner. a) Fluorescence intensity is decreased in a dose-dependent manner at 72 h post-infection with green fluorescent protein-RSV. Fluorescence intensity was quantified using ImageJ 72 h post-infection. Optate significantly decreased RSV infection to the level of the negative control group at doses ⩾50% (n=4, p⩽0.001). b) pH decreases as the dose (buffer content) of Optate decreases. Per a), Optate is most effective at doses >50%, which are above pH 9.2. Dose is defined as percentage of glycine buffer in Optate, with 100% representing 120 mM, 75% representing 90 mM, 50% representing 60 mM, and 25% representing 30 mM. c) Optate pH strongly correlates with intracellular fluorescence intensity in cells treated with pH-sensitive intracellular dye pHrodo Red. ***: p⩽0.001.  Detection of GFP and RSV fusion glycoprotein by ELISA After 5 days of infection with RSV and treatment with either Optate or PBS, growth medium was removed and cells were rinsed in PBS. Cells were then lysed by adding 50 μL of 1X Cell Extraction Buffer PTR (GFP in vitro CatchPoint SimpleStep ELISA) and phosphatase inhibitor, which was directly applied to ALI filters. Cells were scraped off the filters using a flat pipette tip and lysate was transferred to a microfuge tube. Samples were centrifuged at 18 000×g for 20 min at 4°C. The supernatants were transferred into clean tubes and the pellets were discarded. Samples were stored at −80°C until the assay was performed. The sample protein concentration in the extract was quantified using a protein assay (Pierce BCA Protein Assay; Thermo Fisher Scientific). Samples were diluted to desired concentration in 1X Cell Extraction Buffer PTR immediately prior to use. GFP in vitro CatchPoint SimpleStep ELISA (Abcam, Cambridge, UK) was used to quantify GFP and RSV was quantified using RSV (A2) Fusion Glycoprotein (RSV-F) ELISA Kit (SinoBiological, Wayne, PA, USA). Technical replicates were averaged.
Results were plotted on a four-parameter logistic regression model per package instructions, and concentrations were calculated [20].

Immunoblots
Using the same protocol as that for ELISA, cells were lysed in 1X Cell Extraction Buffer PTR. Capillary electrophoresis was performed on the automated Jess system as described previously (ProteinSimple, San Jose, CA, USA) [14]. Briefly, 0.25 μg·μL −1 lysate was plated and run according to the manufacturer's recommendations. Antibodies to GFP ( part number 2956T; Cell Signaling Technology, Boston, MA, USA) were used. Compass software (ProteinSimple) generated digitally rendered bands based on chemiluminescence electrophoretogram to quantify GFP.

Analysis
All statistical analyses were calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and R (R Core Team, Vienna, Austria). To evaluate differences in fluorescence intensity, fluorescent particle counts, viral plaques and RSV-F and GFP protein quantities between negative control and treatment groups, a two-sample two-tailed t-test was used for Gaussian-distributed data and Wilcoxon rank-sum test was used for non-Gaussian-distributed data (supplementary table S1). For cases with more than two groups, robust ANOVA models were considered with pairwise Tukey's test. With multiple time points, the tests were performed for each time point separately. To study the association with pH, robust linear regression models were fitted. For ELISA data, a four-parameter logistic regression model was used [20]. A p-value <0.05 was considered statistically significant.

Biosafety and ethics statement
All experiments were conducted at Indiana University (Indianapolis, IN, USA) in a biosafety level 2 laboratory (IBC# IN-1127). Primary cells were obtained under Indiana University institutional review board protocol #1408855616. All subjects provided informed consent.

Optate treatment inhibits RSV infection in submerged cells
In submerged HAECs, RSV infection was lower in cells treated with Optate compared to control at 48 h and 72 h. Figure 1a shows fluorescence microscopy images taken 72 h post-infection and demonstrates the difference in RSV infection between Optate-treated cells and PBS control. Figure 1b demonstrates a significant decrease in fluorescence intensity quantified using ImageJ during primary infection (48 h n=4, p=0.0174; 72 h n=4, p<0.0001). These results were confirmed by fluorescent particle count and plaque assays from supernatant (48 h n=9, p=0.0178; 72 h n=9, p=0.0019 and 48 h n=7, p=0.0011; 72 h n=7, p=0.0148, respectively; figure 2a and b). Immediately following the incubation period, supernatant collected from Optate-treated cells had a significantly higher viral load indicated by increased fluorescent particle counts (n=7, p=0.0017) and particle-forming units (PFUs) (n=8, p=0.0011) compared to PBS control ( figure 2c and d). Inhibition of RSV by optate is dose-dependent RSV infection in submerged HAECs decreased in a dose-dependent manner, as shown in figure 3. Figure 3a shows the decrease in viral fluorescence intensity quantified using ImageJ. Optate significantly decreased RSV infection to the level of the negative control group at doses ⩾50% (n=4, p<0.001). Furthermore, pH decreased as the dose of Optate decreased (figure 3b). Finally, Optate pH strongly correlated with intracellular fluorescence intensity in cells treated with pH-sensitive intracellular dye pHrodo Red (R 2 =0.8406, figure 3c).

Optate treatment inhibits RSV infection in organotypic, differentiated primary HAECs
In primary HAECs cultured at ALI, fluorescence intensity decreased in cells treated with Optate compared to control during primary infection (p⩽0.05; figure 4a, b and c). Cells were treated twice daily with Optate or PBS control and fluorescent images were obtained every 24 h post-infection. In addition, there was a significant decrease in the amount of GFP quantified using Jess (figure 5a and b) and ELISA (figure 5c), along with a significant reduction in RSV-F protein quantified using ELISA ( figure 6).

Discussion
These experiments demonstrate that Optate inhibits RSV infection and replication in primary HAECs grown under both submerged conditions and at ALI. Furthermore, RSV infection decreased with increasingly concentrated Optate administration. Our group has shown previously that treatment with Optate raises both intracellular and extracellular pH, is not cytotoxic, is safe and well tolerated in humans, and prevents SARS-CoV-2 replication in primary HAECs (supplementary video S1) [14,15].
Of note, fluorescent particle count and PFUs in the plaque assays of supernatant from Optate-treated cells increased immediately after RSV inoculation ( figure 2c and d), despite the fact that Optate reduced intracellular RSV infection and replication at later time points (figure 2a and b). This suggests decreased uptake of the virus into the cells. Altered endosomal trafficking caused by Optate may explain the decrease in viral entry into the cell [14]. At later time points, a decrease in fluorescent particle count and PFUs in plaques assays of Optate-treated cells suggests that Optate also inhibits RSV replication within the cell. Figure 7 illustrates our proposed mechanisms for RSV inhibition by Optate.
Our study has several limitations. All the data presented from our cell culture models in vitro may not translate to in vivo systems and disease processes. In addition, our use of fluorescence microscopy to quantify infection may be confounded by the presence of multiple layers of cells in ALI. Furthermore, our current results support the hypothesis that Optate inhibits RSV through alterations in endosomal trafficking, but further studies are needed to confirm this mechanism. One of the challenges of studying airway pH is that exact measurements of pH are difficult to obtain and require direct sampling during bronchoscopy. Even then, the act of sampling airway pH may affect the results. Exhaled breath condensate pH and changes in exhaled nitric oxide are therefore often used as surrogate markers of airway pH. Although a normal range of pH has been established a using exhaled breath condensate, exact airway pH measurements are unknown. Using exhaled breath condensate pH and changes in exhaled nitric oxide, we know that Optate transiently raises airway pH in humans in vivo, but to what extent is unknown [15].
In conclusion, Optate reduces RSV infection in primary HAECs. Our results suggest that this is due to increased intracellular pH [14]. These findings suggest that Optate might be the focus of additional studies aimed at developing a treatment for patients with RSV. These studies could include further mechanistic experiments in addition to clinical studies.