OM-85 Broncho-Vaxom®, a Bacterial Lysate, Reduces SARS-CoV-2 Binding Proteins on Human Bronchial Epithelial Cells

In clinical studies, OM-85 Broncho-Vaxom®, a bacterial lysate, reduced viral respiratory tract infection. Infection of epithelial cells by SARS-CoV-2 depends on the interaction of its spike-protein (S-protein) with host cell membrane proteins. In this study, we investigated the effect of OM-85 on the expression of S-protein binding proteins by human bronchial epithelial cells. Human bronchial epithelial cells were treated with OM-85 over 5 days. The expression of SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2), transmembrane protease serine subtype 2 (TMPRSS2), dipeptidyl peptidase-4 (DPP4), and a disintegrin and metalloprotease 17 (ADAM17) were determined by Western blotting and quantitative RT-PCR. Soluble (s)ACE2, heparan sulfate, heparanase, and hyaluronic acid were assessed by ELISA. OM-85 significantly reduced the expression of ACE2 (p < 0.001), TMPRSS2 (p < 0.001), DPP4 (p < 0.005), and cellular heparan sulfate (p < 0.01), while ADAM17 (p < 0.02) expression was significantly upregulated. Furthermore, OM-85 increased the level of sACE2 (p < 0.05), hyaluronic acid (p < 0.002), and hyaluronan synthase 1 (p < 0.01). Consequently, the infection by a SARS-CoV-2 spike protein pseudo-typed lentivirus was reduced in cells pretreated with OM-85. All effects of OM-85 were concentration- and time-dependent. The results suggest that OM-85 might reduce the binding of SARS-CoV-2 S-protein to epithelial cells by modification of host cell membrane proteins and specific glycosaminoglycans. Thus, OM-85 might be considered as an add-on for COVID-19 therapy.


Introduction
Epithelial cells of the oral cavity, nasal duct, and upper airway are the primary targets for SARS-CoV-2 [1][2][3], by the interaction of the viral spike protein (S-protein) with the human angiotensin converting enzyme 2 (ACE2) and other host proteins and glycosaminoglycans [4,5]. Moreover, a recent study defined the susceptibility to SARS-CoV-2 infection as the result of a combination between the genetic variations of SARS-CoV-2 and the genetics of the host [6]. This implies that the different genetic variants of the viral S-protein bind with different strength to ACE2 variants, and this might play a role in the severity of infection.
ACE2 is constitutively expressed on the cell membrane of respiratory tract epithelial cells and helps the SARS-CoV-2 to bind and infect. Treatment with human recombinant soluble ACE2 (sACE2) has been suggested as a scavenger therapy to trap SARS-CoV-2 before binding to the mACE2 [7]. The generation of sACE2 is mainly controlled by the activity of ADAM17 (a disintergrin and metalloproteinase 17), which is also expressed on the membrane of epithelial cells [8,9]. ADAM17 expression is sensitive to viral infection including influenza and SARS-CoV-2 [8,10]. However, there is no direct evidence that recombinant ACE2 binds and neutralizes SARS-CoV-2. Patients with hypertension, diabetes, or obesity present the major risk group for COVID-19 and are characterized by high levels

Cell Lines
Two immortalized human bronchial epithelial cell lines (BEAS-2B and Nuli) were purchased from ATCC (Manassas, VA, USA). Four primary epithelial cell lines were established from endobronchial biopsies of patients undergoing bronchoscopy for diagnostic reason. All patients gave written informed consent, and the procedure was approved by the local ethical committee (PB_2019-00035). Epithelial cells were grown to confluence in CnT-PR-A (CellnTech, Bern, Switzerland). Epithelial cells were characterized for positive expression of E-cadherin and/or cytokeratin-14, and negative staining for α-smooth muscle actin, as described earlier [31].
Confluent cells were treated with different dilutions of OM-85 (0, 1:10, 1:20, 1:50, and 1:100) in cell culture medium. Two different treatment schemes were applied: (i) a single treatment on Day 0 in order to study the duration of the OM-85 induced effect on protein expression and (ii) daily refreshment of diluted OM-85 to mimic the daily application in patients ( Figure 1).

Protein Analysis
Cellular membrane proteins were collected in a Mem-PER (Pierce, Rockford, IL, USA). The protein concentration was determined by BCA (Thermo Fisher Scientific, Basel, Switzerland) and equal amounts of protein (10 µ g) per sample were size fractionated in a gradient PAGE-gel (8-12%, BioRad, Basel, Switzerland), transferred to nitrocellulose membrane and incubated for 1 h in PBS + 2% BSA before being incubated overnight with an antibody specific to one of the target proteins (Table 1). Protein bands were visualized after washing and incubation with a secondary HRP labelled antibody using the AZURE imaging system, as described earlier [31].

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Messenger RNA was extracted from confluent cells after treatment using a Trizolfree extraction kit (Zymo Research, Tustin, CA, USA). Total RNA concentration was adjusted to 1 µ g/µ L, and qRT-PCR was performed using an ABI-7500 system (Applied Biosystem, Thermo Fisher Scientific, Allschwil, Switzerland) using the primers listed in Table 2. The PCR conditions were as follows: melting for 10 min at 95 °C, followed by up to 45 cycles consisting of 30 s at 95 °C and 1 min at 60 °C. The expression level of mRNAs was assessed for ACE2, TMPRSS2, DPP4, and ADAM17.

Protein Analysis
Cellular membrane proteins were collected in a Mem-PER (Pierce, Rockford, IL, USA). The protein concentration was determined by BCA (Thermo Fisher Scientific, Basel, Switzerland) and equal amounts of protein (10 µg) per sample were size fractionated in a gradient PAGE-gel (8-12%, BioRad, Basel, Switzerland), transferred to nitrocellulose membrane and incubated for 1 h in PBS + 2% BSA before being incubated overnight with an antibody specific to one of the target proteins (Table 1). Protein bands were visualized after washing and incubation with a secondary HRP labelled antibody using the AZURE imaging system, as described earlier [31].

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Messenger RNA was extracted from confluent cells after treatment using a Trizol-free extraction kit (Zymo Research, Tustin, CA, USA). Total RNA concentration was adjusted to 1 µg/µL, and qRT-PCR was performed using an ABI-7500 system (Applied Biosystem, Thermo Fisher Scientific, Allschwil, Switzerland) using the primers listed in Table 2. The PCR conditions were as follows: melting for 10 min at 95 • C, followed by up to 45 cycles consisting of 30 s at 95 • C and 1 min at 60 • C. The expression level of mRNAs was assessed for ACE2, TMPRSS2, DPP4, and ADAM17.

Soluble ACE2 (sACE2) and Glycosaminoglycans
ACE2 and glycosaminoglycans were determined in cell culture medium by enzymelinked immuno-sorbent assay (ELISA), in accordance with the instructions of the manufacturer. The sACE2 kit was obtained from Abcam (Cambridge, UK), and the glycosaminoglycan kit from antibodies-online (Aachen, Germany).  On Day 3, the cells were infected with 0.1 MOI of pseudo-type lentivirus for 30 min. The cells were washed with medium twice, before being incubated in standard cell culture condition (37 • C, 5% CO 2 , 100% humidity) for 24 and 48 h. None-infected cells were used as a control for unspecific background green fluorescence. The infection status was determined by fluorescence microscopy and image analysis for signal intensity.

Statistics
The null hypothesis was that OM-85 did not affect the mRNA expression, nor the protein expression of ACE2 and its regulating proteins. Data were compared by ANOVA and Student's t-test; p < 0.05 were considered significant and data are expressed as mean ± SEM.

Results
The expression of the SARS-CoV-2 interacting human host proteins (ACE2, TMPRSS2, DPP4, and ADAM17) were analyzed in two immortalized human bronchial epithelial cell lines (BEAS-2B and Nuli) and four primary human bronchial epithelial cells isolated from patients without chronic inflammatory lung diseases. The immortalized cells were compared to the primary cells, showing there was no significant difference regarding the overall response to OM-85 treatment. Therefore, we combined the results for each protein for all epithelial cell lines in the following sections

OM-85 Regulates SARS-CoV-2 Interacting Proteins on the Protein Level
All epithelial cells expressed mACE2, TMPRSS2, DPP4, and ADAM17. The single treatment strategy with OM-85 on Day 0 had no significant effect on the expression level of ACE2 (Supplementary Figure S1). Therefore, all subsequent data were obtained in cells treated with daily application of OM-85.
First, the expression of ACE2 and the proteins that might affect its expression were as- OM-85 also significantly reduced the expression level of TMPRSS2 mRNA on both days ( Figure 2B). The expression of DPP4 encoding RNA was significantly reduced in OM-85-treated cells, only after 48 h ( Figure 2C). In contrast to all other mRNAs, the level of ADAM17 mRNA was significantly increased by OM-85 treatment after 48 h ( Figure 2D).
Biomedicines 2021, 9,1544 response of ACE2 protein to OM-85 was concentration dependent and only b significant at dilutions of 1:50 and 1:10 on Day 5 with daily repeated treat (Supplementary Figure S2). OM-85 also significantly reduced the expression le TMPRSS2 mRNA on both days ( Figure 2B). The expression of DPP4 encoding RN significantly reduced in OM-85-treated cells, only after 48 h ( Figure 2C). In contras other mRNAs, the level of ADAM17 mRNA was significantly increased by O treatment after 48 h ( Figure 2D).  A kinetic study of protein expression for ACE2, TMPRSS2, DPP4, and ADAM17 by epithelial cells undergoing daily OM-85 treatment over 4 days is shown in Figure 2E. Image analysis shows that OM-85 significantly reduced ACE2 protein expression at all time points ( Figure 2F). The reducing effect of OM-85 on TMPRSS2 protein expression became significant only after 4 days ( Figure 2G). A similar effect was seen for DPP4 levels, which significantly declined after 24 h, reached control level on Day 2 and Day 3, and significantly declined on Day 4 ( Figure 2H). This pattern of up and down for DPP4 protein was seen in all cell lines. In contrast, the expression of ADAM17 protein significantly increased at all time points, as shown in Figure 2I.

The Generation of Soluble ACE2 (sACE2)
The generation of sACE2 by human bronchial epithelial cells was stimulated by OM-85 over 5 days, as determined in the cell culture medium by ELISA ( Figure 3A). The concentration of sACE2 in OM-85-treated cells increased at a faster rate as compared with untreated cells and became significant on Day 4, as shown in Figure 3A. The stimulating effect of OM-85 on sACE2 was concentration dependent and significant on Days 4 and 5 ( Figure 3B). Comparing the concentration of sACE2 with the concentration of ADAM17, as reported above, showed a linear positive correlation with a R 2 value of 0.8686 ( Figure 3C). A kinetic study of protein expression for ACE2, TMPRSS2, DPP4, and ADAM17 by epithelial cells undergoing daily OM-85 treatment over 4 days is shown in Figure 2E. Image analysis shows that OM-85 significantly reduced ACE2 protein expression at all time points ( Figure 2F). The reducing effect of OM-85 on TMPRSS2 protein expression became significant only after 4 days ( Figure 2G). A similar effect was seen for DPP4 levels, which significantly declined after 24 h, reached control level on Day 2 and Day 3, and significantly declined on Day 4 ( Figure 2H). This pattern of up and down for DPP4 protein was seen in all cell lines. In contrast, the expression of ADAM17 protein significantly increased at all time points, as shown in Figure 2I.

The Generation of Soluble ACE2 (sACE2)
The generation of sACE2 by human bronchial epithelial cells was stimulated by OM-85 over 5 days, as determined in the cell culture medium by ELISA ( Figure 3A). The concentration of sACE2 in OM-85-treated cells increased at a faster rate as compared with untreated cells and became significant on Day 4, as shown in Figure 3A. The stimulating effect of OM-85 on sACE2 was concentration dependent and significant on Days 4 and 5 ( Figure 3B). Comparing the concentration of sACE2 with the concentration of ADAM17, as reported above, showed a linear positive correlation with a R 2 value of 0.8686 ( Figure  3C).

OM-85 Reduced HS by Increasing Heparanase Expression, but Increased HA Secretion
Epithelial cells produced cellular HS, as well as soluble HS (sHS), and heparanase. OM-85 treatment increased the concentration of heparanase, which became significant after 3 days for a dilution of 1:50 and Day 5 for a dilution of 1:100 ( Figure 4A). sHS was also increased by OM-85 treatment and became significant only for dilution 1:50 on Days 3 and 4 ( Figure 4B). The effect of OM-85 on cellular HS was determined in membrane proteins by ELISA, and showed a significant dilution-dependent decrease on Days 3 and 4 ( Figure 4C). However, the reducing effect of OM-85 showed a large variation between the different cell lines. sHS correlated with heparanase concentration (R 2 : 0.965), as shown in Figure 4D.  The synthesis and secretion of HA was significantly stimulated by OM-85 (1:50) after 24 and 48 h as compared with untreated cells at the same time points ( Figure 5A). This effect correlated with the increased expression of hyaluronan synthase 1 (Has-1) after 24 h in the presence of OM-85 ( Figure 5B). In contrast, OM-85 had no stimulating effect on the expression of Has-2 and Has-3 ( Figure 5C,D). In unstimulated cells, the secretion of hyaluronidase 1 (Hyal-1) increased over 24 h, which was prevented by OM-85 treatment ( Figure 5E). A similar effect of OM-85 was observed for Hyal-2 ( Figure 5F). The synthesis and secretion of HA was significantly stimulated by OM-85 (1:50) after 24 and 48 h as compared with untreated cells at the same time points ( Figure 5A). This effect correlated with the increased expression of hyaluronan synthase 1 (Has-1) after 24 h in the presence of OM-85 ( Figure 5B). In contrast, OM-85 had no stimulating effect on the expression of Has-2 and Has-3 ( Figure 5C,D). In unstimulated cells, the secretion of hyaluronidase 1 (Hyal-1) increased over 24 h, which was prevented by OM-85 treatment ( Figure 5E). A similar effect of OM-85 was observed for Hyal-2 ( Figure 5F).

OM-85 Pretreatment Reduces Infection of Epithelial Cells with Pseudo-Typed S-Protein Lentivirus
The above presented results suggest that OM-85 might have the potential to reduce the binding of SARS-CoV-2 to epithelial cells. In order to verify such an effect, a pseudotyped lentivirus expressing the SARS-CoV-2 S-protein, as well as carrying a green fluorescence protein was used to mimic the infection of human epithelial cells with SARS-CoV-2. As shown in Figure 6, the green fluorescence indicating infection was significantly reduced in cells pretreated with OM-85 for 24 h. This effect correlated with the dilution of OM-85 (1:20, 1:50, and 1:100), as well as over time ( Figure 6A,B). The effect of OM-85 on pseudo-typed lentivirus infection was calculated from the number of green fluorescence cells in two cell lines, in an area of 40,000 µm 2 and at three locations on the same slide. The analysis of the data showed that OM-85 had a concentration-dependent preventive effect on infection on both days ( Figure 6C,D).

OM-85 Pretreatment Reduces Infection of Epithelial Cells with Pseudo-Typed S-Protein Lentivirus
The above presented results suggest that OM-85 might have the potential to reduce the binding of SARS-CoV-2 to epithelial cells. In order to verify such an effect, a pseudotyped lentivirus expressing the SARS-CoV-2 S-protein, as well as carrying a green fluorescence protein was used to mimic the infection of human epithelial cells with SARS-CoV-2. As shown in Figure 6, the green fluorescence indicating infection was significantly reduced in cells pretreated with OM-85 for 24 h. This effect correlated with the dilution of OM-85 (1:20, 1:50, and 1:100), as well as over time ( Figure 6A,B). The effect of OM-85 on pseudotyped lentivirus infection was calculated from the number of green fluorescence cells in two cell lines, in an area of 40,000 µm 2 and at three locations on the same slide. The analysis of the data showed that OM-85 had a concentration-dependent preventive effect on infection on both days ( Figure 6C,D).

Discussion
The above presented data indicate that daily application of OM-85 reduces the expression of mACE2 and other cell membrane proteins, which play a role in the attachment of SARS-CoV-2 and the infection of human epithelial cells. The results further imply that the reduced expression of mACE2 in OM-85-treated cells is potentially mediated through an increase in ADAM17. Furthermore, OM-85 reduced the expression of HS, which is another component necessary for SARS-CoV-2 infection. In contrast, OM-85 increased the synthesis of HA, which may protect against SARS-CoV-2 infection. All of these effects of OM-85 on epithelial cell membrane proteins and GAGs might explain the reduced infection of epithelial cells with the pseudo-type lentivirus expression SARS-CoV-2 S-protein.

Discussion
The above presented data indicate that daily application of OM-85 reduces the expression of mACE2 and other cell membrane proteins, which play a role in the attachment of SARS-CoV-2 and the infection of human epithelial cells. The results further imply that the reduced expression of mACE2 in OM-85-treated cells is potentially mediated through an increase in ADAM17. Furthermore, OM-85 reduced the expression of HS, which is another component necessary for SARS-CoV-2 infection. In contrast, OM-85 increased the synthesis of HA, which may protect against SARS-CoV-2 infection. All of these effects of OM-85 on epithelial cell membrane proteins and GAGs might explain the reduced infection of epithelial cells with the pseudo-type lentivirus expression SARS-CoV-2 S-protein.
In clinical studies, OM-85 reduced the risk of viral lower respiratory tract infections other than SARS-CoV-2 [33][34][35][36][37]. In a randomized placebo controlled double-blinded study, it was shown that OM-85 treatment over 10 consecutive days significantly reduced the chance of recurrent respiratory tract infections [33]. In a second randomized trial, OM-85 prevented recurrent respiratory tract infection in very young children significantly [34]. When OM-85 was combined with inhaled steroids, the number of recurrent respiratory tract infections and the severity of asthma attacks was significantly reduced in a cohort of 60 children with asthma [36]. This is of interest because steroid treatment has been reported to increase the risk of infection [38,39]. A study by Lu et al. [36] suggested that OM-85 might increase the anti-inflammatory effect of steroids by increasing interferon-γ, but decreasing pro-inflammatory IL-4 and IL-10. In a meta-analysis of efficacy and safety in children, it was suggested that OM-85 might have a protective effect on COVID-19 [32].
Furthermore, OM-85 therapy significantly reduced the number of exacerbations and respiratory tract infections in patients with allergic rhinitis, asthma, or COPD [35]. A metaanalysis including 1190 COPD patients treated with OM-85 revealed a 39% reduction of exacerbations [37]. The same study found that OM-85 had no effect on the length of hospitalization for COPD patients. These findings suggest that the anti-viral effect of OM-85 is not virus specific, but improves the overall host defense against viral infection.
In the context of COVID19 therapy, steroids were among the first drugs to reduce severity, and the cytokine storm by independent mechanisms: (i) The anti-inflammatory effect of steroids prevented the cytokine storm [40,41]; (ii) ACE2 expression was reduced by inhaled steroids, which reduced the chance of SARS-CoV-2 to infect epithelial cells [42]; (iii) dexamethasone directly blocked the interaction of the SARS-CoV-2 spike protein with ACE2 [43]; (iv) steroids increased the activity of ACE2 in patients, which might have contributed to the organ protective effects of ACE2 [44]. In non-intubated severe COVID-19 patients, methylprednisolone reduced the need of mechanical ventilation [45,46].
ACE2 is the key host protein that enables SARS-CoV-2 to infect human cells [7]. It had been suggested that reducing ACE2 might prevent SARS-CoV-2 infection, which has been confirmed in vitro [15]. The reduction of mACE2 in OM-85-treated epithelial cells inversely correlated with the concentration of sACE2 in the cell culture medium. Considering that the mRNA level of ACE2 was not significantly reduced by OM-85, this suggests that OM-85 activates the shedding of ACE2. Interestingly, steroids also reduce the expression of ACE2, which might partly explain the beneficial effects of steroids in COVID-19 patients [42,47]. Thus, the observed reduced expression of mACE2 after daily OM-85 treatment of epithelial cells might reduce the susceptibility to be infected with SARS-CoV-2.
To date, only one enzyme has been identified to cleave ACE2 from the cell membrane, i.e., ADAM17 [8]. The expression of ADAM17 by human bronchial epithelial cells was confirmed by our data, and furthermore, it was upregulated on the mRNA and protein level by OM-85. Analyzing the correlation between sACE2 and ADAM17, the data implies that OM-85 reduces membrane ACE2 by increasing ADAM17 expression. A similar mechanism was reported for viruses leading to the increased cleavage of ACE2 via increased ADAM17 [10].
As shown above, OM-85 upregulated the expression of HA and Has-1. The upregulation of Has-1 suggests that OM-85 supports the de novo synthesis of long-chain HA, which has been described to reduce viral infections [48,49]. In this context, SARS-CoV-2 binding to the host cell was reduced by HA and other GAGs [50][51][52]. Thus, in addition to the reduced expression of mACE2, this increased expression of HA by cells treated with OM-85 might further reduce SARS-CoV-2 infection.
HS supports infection of epithelial cells by SARS-CoV-2 [53]. Therefore, reducing HS on cell surfaces was suggested as an anti-rival therapeutic strategy. Three recent studies implied that HS also facilitates the binding of SARS-CoV-2 to host cells, and therefore is part of COVID-19 [54][55][56][57]. In the cell culture medium of OM-85-treated epithelial cells, the level of cellular HS was significantly reduced as compared with untreated cells. The reduction of cellular HS is most likely caused by an increase of heparanase in OM-85-treated cells, which was correlated with an increase in soluble HS. However, the role of HS as a target in SARS-CoV-2 therapy is controversially. In contrast to the above cited studies, others have suggested that it would be beneficial to upregulate the expression of HS, and thereby block the infection of epithelial cells with SARS-CoV-2 [53,58]. Two studies suggested that soluble HS might interact with the virus, and thereby prevent its binding to cell surface HS, which inconsequence, would reduce SARS-CoV-2 infection of epithelial cells [59,60].
Despite the presented experimental data, there are some limitations in this study which include: (i) The lack of cellular signaling mechanism to explain the effect of OM-85 on the regulation of SARS-CoV-2 binding host proteins and GAGs. The fact that OM-85 presents a mixture of bacterial proteins from eight different bacteria, makes it difficult to link epithelial cell response to a specific component. (ii) The observation that a single application of OM-85 did not change the expression of all investigated proteins suggests an indirect effect of the compound on those SARS-CoV-2 interacting host proteins that were modified after more than 3 days. (iii) The lack of direct proof that OM-85 reduces SARS-CoV-2 infection due to restricted access to the virus. (iv) In vivo studies in animals or clinical studies will be needed to clarify if the results obtained with the pseudo-typed lentivirus reflects the effect of OM-85 on SARS-CoV-2 infection. (v) The presented data show that the effect of OM-85 on SARS-CoV-2 binding molecules required daily treatment over up to 5 days. This is in line with the instructions from the manufacturer to patients (1 capsule daily over 10 consecutive days per month for 3 months). Incubation periods longer than 5 days caused changes of the cell morphology, and therefore could not be performed.
In conclusion, our data support the hypothesis that OM-85 might help to prevent SARS-CoV-2 infection or reduce COVID-19 severity by lowering the expression of cellmembrane ACE2 and HS, while increasing circulating sACE2 and HA. Data Availability Statement: The data will be available through the corresponding author upon request.