Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

  1. Ravi Kant
  2. Lauri Kareinen
  3. Ravi Ojha
  4. Tomas Strandin
  5. Saber Hassan Saber
  6. Angelina Lesnikova
  7. Suvi Kuivanen
  8. Tarja Sirnonen
  9. Merja Joensuu
  10. Olli Vapalahti
  11. Tom Kirchhausen
  12. Anja Kipar
  13. Giuseppe Balistreri

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Abstract

Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available.

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    Reviewer #1 (Evidence, reproducibility, and clarity):

    Summary: In this work, Kant and co-workers describe a two drugs regimen for therapeutics treatment of SARS-CoV-2 infection. SARS-CoV-2 infection of cells is dependent on the cleavage of the spike S protein by cellular proteases that prime S allowing the envelop protein to fuse of host membrane during entry and delivery of the viral genome to the target cell. The most important cellular protease is TMPRSS2 located at the surface of the cell. However, in cells with low TMPRSS2 levels, Cathepsins, located in endosomes have been shown to be able to also prime S. The therapeutic strategy of the authors relies on the combined usage of an inhibitor of TMPRSS2 (nafamostat) together with a compound that impairs endosomal maturation (apilimod) which is a key step for the activation of cathepsin. The rationale is that a dual regimen would be more effective to inhibit SARS-COV-2 infection. Using cell lines and a combination of SARS-CoV2 infection and pseudotyped VSV particles (VSV virus where the glycoprotein has been replaced by the SARS-CoV-2 spike proteins), the authors could show that a two-drug regimen was more efficient in preventing SARS-CoV-2 infection compared to single drug regimen. The authors next employed a mouse model of SARS-CoV-2 infection and similarly could show that bi-therapy was more efficient in preventing infection. Importantly, the authors describe a new formulation of the drugs that improve stability of the compounds and shelve life which could be of great benefit with respect to storage needs in therapeutic setting of the population.

    While the reviewer thinks the work is potentially very relevant, some of the conclusions are not fully supported by the data and additional experiments/quantifications should be performed to improve rigor and fully support the author conclusions.

    Major comments:

    • Throughout the paper, statistical analysis of the results should be performed to support the conclusion of the authors. Currently many experiments do not have statistical analysis and P values or statical significance are missing in most of the figures: Figure 1B, 1D, 4A, 5B, and S2. RESPONSE: As requested by the reviewer, the results of the statistical analysis of the differences are now reported for Figures 1B, 1D, 4A, 5B, and S2. There is no change in our conclusions as first reported in the original manuscript.

    • Quantification of the various pathology observed in mice should be quantified and scored. In the current version, the authors provided a supplementary table describing the pathology observed in individual mice upon SARS-CoV-2 infection. Adapted scoring of the different pathologies should be performed to obtain a statistical view of the pathology induced by SARS-CoV-2 and how this is prevented by the mono and bi-therapy approaches. RESPONSE: The mouse model employed in the present study, i.e. SARS-CoV-2 Beta infection in BALB/c mice, is characterised by a limited and short-lived viral infection of the lungs and rather subtle pathological changes, as described in detail in our previous publication (Kant et al., 2021. Viruses).

    We chose this model because it better mimics the typical (short-lived) respiratory infection observed in human patients than the K18-hACE2 model where infection is detected in nasal mucosa and lung parenchyma, generally sparing the respiratory epithelium, but also spreads to the brain (Seehusen et al., Viruses, 2022; De Neck et al., Viruses, 2023).

    In our model, infection of the lungs (i.e., alveoli) occurs strictly in association with infection of the airways, including the tracheal, bronchial, and bronchiolar epithelium, like the in hamster model. Pulmonary infection is, however, short-lived and wanes off around day 4. The histopathological changes, i.e. degenerative changes, and an inflammatory response, are at best mild in the untreated mice and not observed at all in successfully treated mice. (as summarized for each individual animal in Supplementary Table 2). . For these reasons, this information cannot be quantified by morphometry (which would be the most objective, hence best approach) or scored (a more subjective approach that would only be valid with distinct quantitative differences).

    Nevertheless, and in agreement with the reviewer that a quantitative approach is useful where possible we provide results from morphometry and to confirm the reduction in the degree of tissue damage (i.e., the extent of apoptotic death of infected respiratory epithelial cells; see comment below).

    • Additionally, table 1, is very difficult to read as mice are classified in 3 experiments but this does not match with the individual figures, making it very hard to look for the phenotypes. Is it an order issue within the table or are murine infection experiments performed in the order described in table 1? In this case, can the data be compared between the experiments as some conditions belong to experiment 2 and other to experiment 3? Given the low number of mice, do the experiments have statistical power? RESPONSE: We agree with the reviewer’s assessment of the figure and have therefore modified the graphs in Figure 2 B, to specifically relate experiments and data, by using circles for Experiment set 1 and squares for Experiment set 2.

    We can confirm the reported results have statistical power, particularly important given the constrain due to the low number of animals we were limited to use. As noted in the figure legends, that now includes the results from the statistical analysis, each of the three experiments included at least three control infected mice treated with vehicle. The infection levels in all the control vehicle treated infected mice are very similar in all three experiments.

    • To show that treatment of mice at 3 or 6 hpi indeed reduce the number of clv-capsase3 positive cells, the authors should perform a complete quantification and not limit their analysis to one representative tissue section from one animal. RESPONSE: Following the reviewer’s recommendation, we have now taken a quantitative approach in addition to illustrating the difference in cleaved caspase-3 expression. We have kept the images that illustrate the effect in tissue sections (Figure 4C).

    Briefly, we compared the extent of viral NP and cleaved caspase-3 expression between lungs of vehicle treated mice and mice treated with the drugs from 6 hpi onwards (3 mice per condition), using morphometry. Indeed, there was no significant difference in the extent of viral antigen NP expression in the lungs of the two groups of mice (Figure 4 B and C), which supports the PCR results representing viral RNA levels (Fig. Figure 4 A). However, there was a significant difference in the extent of cleaved caspase-3 expression in the consecutive sections. The results are shown in the new Figure 4D.

    • the authors insist on the new formulation that improves drug stability. To make this statement, this will need to be actively tested both in cell culture and in animal models: currently, the authors test the drugs stored 3 months at 25c or -20c and show that they remain active, but in this experiment freshly made drug was not directly tested in parallel. RESPONSE: As requested by the reviewer, we have extended our tests, and confirm our original view that the new formulation improves drug stability. Now shown in revised Figure 1C and D, we found equivalent inhibition in the cell infection assay using freshly made drugs and drugs stored at room temperature for 2 months.

    • Additionally, to make such a statement, different concentration of the drugs should be tested to calculate a IC50 for freshly prepared drug and stored drugs (as the current concentration tested might be at saturating concentration). RESPONSE: As requested by the reviewer, we have determined the IC50 for infection in cells of the drugs freshly prepared or stored. As reported in the revised Figure 1D, there were no differences detected.

    • Finally, the mouse experiments are performed with freshly made compounds and if the authors want to highlight the new formulation and increased stability, experiments in mice should be performed also with stored compounds. RESPONSE: We respectfully disagree with the reviewer on the need to perform additional in vivo experiments. We find no differences in the IC50 antiviral activity of the drugs prepared with our formulation and tested with cells in culture, whether fresh or kept for up to 2 months at room temperature. Given these observations, we feel that we cannot justify further animal experiments, neither ethically nor financially, using the same drugs with the same ab initio antiviral activity.

    • Alternatively, statement on drug stability should be removed or strongly tuned down from text. RESPONSE: We believe that the updated information included in the revised manuscript showing no difference in the IC50s of the compounds freshly prepared and stored at room temperature fully supports our original statement.

    • Statistical analysis on figure 2b should be done between Nafamostat alone and dual treatment to show that both drugs are cooperative in term of antiviral activities RESPONSE: We have carried the requested statistical analysis (Figure 2 B and C) and confirm that dual treatment is not only cooperative, but it also shows synergy, as we originally showed in our published work (Kreutzberger et al., Journal of Virology, 2021).

    • The authors state "A quantitative assessment of the *in vivo *synergy is shown here by the enhanced decrease of viral RNA in lungs of mice treated with both drugs at very low concentrations (Figure 2 B, compare using 2 mg/Kg apilimod dimesylate and 4 mg/Kg nafamostat mesylate alone, and in combination)." I guess, the authors want to comment on the fact that 0.2 mg/kg of apilimod and 0.4 mg/kg of nafamostat are as potent as 2 and 4 mg/kg. is that correct? If YES, to make this statement, bi therapy should be compared to mono therapy at the same concentration. RESPONSE: We apologise for not being clearer in the way we presented the information in our original version of the manuscript.

    Briefly, we compared the effect of high and low bi-therapy doses to the effect of Apilimod or Nafamostat used as single drugs at the highest concentrations. When administered alone, high dose Apilimod did not reduce infection. Nafamostat alone, even at 4 mg/Kg, decreases but does not completely block infection. When combined, even at low doses, the two drugs have a stronger antiviral effect than Nafamostat alone (and of course Apilimod, which was ineffective). Importantly, if the combined effect of the two drugs was merely additive, i.e. the arithmetic sum of the single effects, the addition of Apilimod, which alone has no in vivo antiviral activity, would not have improved the effect of Nafamostat. Instead, even at 10 times lower doses, the bi therapy significantly outperformed the single drug Nafamostat. Thus, the effect is synergistic (i.e. the effect of combined drugs is stronger than the mere sum of effects of each single drug).

    • when drugs are injected after infection (Fig 4), the drugs are not active. In fact, unless the reviewer mis-understood the plot, the mouse are even more infected compared to vehicle. The authors wrote that both regimes (3 and 6hpi) are equally less effective compared to drug administered during infection. The authors should write that both regimes are equally non protective. RESPONSE: We thank the reviewer for pointing out this imprecision. The modified text now reads “Both regimes, compared to drug administration at the time of virus inoculation, were equally ineffective in reducing the viral RNA load and NP expression in lungs as determined at 48 h.p.i. (Figure 4A, B).” (Line 236-238).

    • If drugs are not active after infection, does this approach really represent a therapeutic solution. The authors suggest that it does by limiting pathologies, but this needs to be better quantified (see comment above). RESPONSE: Our results suggest that application of the drugs post infection reduced the cytopathic effect of the virus in the respiratory epithelium in the lungs, reflected by a reduced extent of apoptotic cell death in association with infection. The finding is supported by quantitative morphometric analysis as shown in the new Figure 4D (see also comment above).

    • In the rebound experiment: unless the reviewer misunderstood, it appears that no conclusion can be driven from this experiment. Q-PCR data for vehicle animal a 4dpi show no sign of infection, so the experiment is not really interpretable since control animals are no longer positive. The authors suggest that there is less pathologies but this needs to be better quantified (see comment above). RESPONSE: We have tried to better word the rationale and interpretations of this experiment in the text. Following our drug treatments, viral antigen is still present in epithelial cells within the nasal mucosa, we also surmised that a small number of intact virions could have remained attached to the epithelial cells, trapped within their endosomes, or still within the environment surrounding the cells, any of them capable of triggering infection after removal of the drugs. Thus, the rationale behind the rebound experiment was to ask whether such remaining potentially intact virions could lead to a full reinfection of the lung two days after the treatment was stopped - which we found did not.

    We found that the virus did not regain full infectivity once the drug treatment was interrupted, resulting in undetectable lung PCR signal and very limited, sporadic antigen signal in the lung tissue.

    Minor comments:

    • I__t will make reading easier if the authors always mentioned which drugs inhibit what. For example: addition of the TMPRSS2 inhibitor nafamostat etc.... or addition of apilimod to block cathepsins activities.....__ RESPONSE: Done

    • Figure 1: make a comment in the text that cells with low TMPRSS2 are more sensitive to the cathepsin inhibitor apilimod and vice versa, cells with high TMPRSS2 are more sensitive to nafamostat. This is expected and it could be highlighted. RESPONSE: Done

    • Figure 2B: how are the data normalized? should not RdRp, E and SubE all have a mean at 100% for the vehicle? RESPONSE: Done. Data are now normalized to the mean of RdRp measurements (which is indicated as 100%).

    • Line 211: something is missing here "when (Fig 2...) RESPONSE: Corrected

    • Line 221 should figure 4c RESPONSE: Corrected

    • Figure legends should only contain the details of the experimental design but should not contain description and interpretation of data. This is very minor and maybe a question of taste. __RESPONSE: __ Our figure legends are descriptive for some results and are in accordance with the style of PLOS Pathogens, the journal we are aiming this study.

    *Editorial note: *

    Referees cross-commenting: The other reviewers have highlighted the same limitations concerning the lack of quantifications of the immunochemistry and also the lack of robust statistical analyses. This should be highlighted to the authors as it appears to be the minimum to do prior publication. This should not take too much time as the data are in principle already available

    Reviewer #1 (Significance):

    The work by Kant and co-workers is potentially very significant but some limitations (as highlighted above) impair the impact of the work in his current version. The approach employing a two-drug regimen to combat SARS-COV-2 infection by targeting both TMPRSS2 and cathepsin activities is not new and was described before by the authors themselves. Employing this approach in an animal model is new and the new formulation improving drug stability and facilitating storage could be a game changer in therapeutic setting of patients. As such, this work could be highly significant and of broad interest. However, additional experiments and clarifications are needs to elevate this work to high impact standards. The reviewer believes that the requested experiments are easily achievable by the research teams of this project and think that the project will ultimately have a strong impact in the field.

    Reviewer #2 (Evidence, reproducibility and clarity):

    In this paper, the authors tested the antiviral activity of a combination of compounds by intranasal instillation in a mouse model of SARS-CoV-2. The two compounds used are PIKfyve Kinase inhibitor apilimod dimesylate, which inhibits endosomal maturation, and TMPRSS2 protease inhibitor nafamostat mesylate. The authors have previously shown that a combination of these two inhibitors acts synergistically to prevent entry and infection of SARS-CoV-2 in cell culture. Here, they further investigated the anti-SARS-CoV-2 activity of their combination of compounds by in vivo testing. They used Balb/c mice intranasally inoculated with the Beta variant of SARS-CoV-2. Their data show that concurrent administration of the combo together with the virus prevented lung infection without blocking nasal replication. Delayed administration of the compounds did not reduce replication in the lungs. The only effect was a decrease in bronchiolar cell death. Furthermore, they also tested the stability of the combo at room temperature and their data indicate that these compounds can be kept at room temperature for at least 3 months without losing antiviral activity, at least when resuspended in water. These data are potentially interesting, but they need to be consolidated by additional experiments.

    Major comments:

    • The authors only present immunohistochemistry to investigate viral replication in the nose. A quantitative analysis of replication would allow for better conclusions concerning viral replication in this organ. RESPONSE: We appreciate the reviewer’s comment and the wish to see viral antigen expression quantified in the nasal mucosa. As described below, however, practicalities associated with sample preparation prevented us from performing morphometric analysis. The complementary quantification of viral replication requires viral RNA by PCR. Unfortunately, we had not planned this aspect of the study and therefore did not collect the required fresh samples from nasal turbinates required for this analysis. Although interesting to investigate, we feel this is not vital for reaching the interpretation and conclusions derived from the current study. We thereby don’t think that this would be sufficient reason to undertake another round of infections, particularly taking into consideration that it would require sacrificing another significant number of animals.

    We could extend our morphometric analysis used in the lung and adapt it to the nasal mucosa. However, we are of the opinion that this would not provide trustworthy results. The main reason for this limitation is due to a problem that occurs during decalcification and paraffin embedding of the heads, which results in large variations in the area of the nasal mucosa as well as the olfactory epithelium in each section in different animals (Figure 3C provides some evidence of this).

    Briefly, we cut the entire heads longitudinally in the midline with a diamond saw and then gently decalcify the two halves of the head. This is followed by paraffin embedding. At some point during the process some of the thin and soft bits of nasal mucosa can become twisted and distorted, moving away from the cut surface exposed to the microtome blade. Therefore, the paraffin sections (appr. 3 µm thick) will in their majority not comprise full sections of the nasal mucosa. An objective comparative quantification of the extent of NP expression in the nasal mucosa would require (nearly) the entire mucosa to be assessed.

    • Complementary investigation on a potential anti-inflammatory effect of the drugs would also be welcome. Furthermore, it is surprising that the authors did not report potential weight changes. RESPONSE: Our mouse model, i.e. SARS-CoV-2 Beta infection in BALB/C mice, is characterised by the limited and short-lived viral infection of the lungs, rather subtle pathological changes and a limited inflammatory response strictly associated with the presence of viral antigens, as we previously described (Kant et al., 2021. Viruses). Hence, other animal models (for example the hamster model) would be more appropriate. Though potentially interesting, such investigations are beyond the current scope of our studies.

    In our study, the animal weight did not change during infection, in agreement with our earlier published work with the same animal model (Kant et al., 2021. Viruses). These data is now included in this manuscript.

    • It would have been interesting to complete the experiments with a demonstration that the compounds block viral transmission. RESPONSE: While it would be interesting to see whether the combined drugs also block viral transmission, such an experiment would require the use of a different animal model (possibly hamsters), an endeavour that is beyond the scope of our study. In our experience BALB/C mice infected with SARS-CoV-2 Beta variant do not transmit the virus. We have co-housed naïve BALB/C mice for 4 days with BALB/C mice intranasally challenged with 6 x 10^4 PFU SARS-CoV-2 Beta and have no evidence of virus transmission to the naïve mice (unpublished results). Similar results with C57BL/6 WT mice were obtained by Pan et al., Signal Transduction and Targeted Therapy, 2021).

    Minor comments:

    • The second paragraph of the introduction is not clear. It needs to be re-written. Furthermore, there is no evidence that Calu3 cells do not express cathepsins. RESPONSE: We have clarified this section of the introduction as follows:

    “It has been shown previously that SARS-CoV-2 infection can be blocked by serine protease inhibitors such as nafamostat mesylate in cells that express high levels of TMPRSS2 but very low or undetectable levels of cathepsin B/L (e.g. Calu-3 cells)5-7. In cells that instead express cathepsins but not TMPRSS2 (e.g. VeroE6 or A549 cells), infection depends on the delivery of endocytosed viruses to endo/lysosomes, a process that can be efficiently inhibited by drugs that interfere with endosome maturation and acidification such as Bafilomycin A1, chloroquine or ammonium chloride”.

    • Figure 4C: Is there any explanation for the lack of apoptosis? The authors should at least provide some hypotheses. Furthermore, this figure is quoted as Figure 4B in the text instead of Figure 4C. RESPONSE: For the revised manuscript, we have quantified the extent of apoptosis by a morphometric analysis of cleaved caspase-3 expression in the lung sections (now provided in new Figure 4C).

    We presently do not have an explanation for the reduction in the cytopathic effect of the virus, particularly in respiratory epithelial cells. This is an area of research we plan to continue investigating in future. We have commented on this in the Discussion session of the revised manuscript (Line 301-307).

    • Line 199: The authors claim that the effect of their combo is synergistic. However, this cannot be clearly concluded without appropriate additional experiments where they vary the concentration of the compounds. RESPONSE: The work we report here with mice is a follow up of our earlier work demonstrating the antiviral synergy of nafamostat and apilimod with cells in culture (Kreutzberger et al., Journal of virology, 2021). See comments to Reviewer 1.

    • Line 211: The sentence is incomplete RESPONSE: Fixed.

    • The lettering in the panels needs to be doublechecked. RESPONSE: Done.

    Reviewer #2 (Significance):

    __General assessment: __Finding new antiviral against SARS-CoV-2 remains a priority to fight against COVID-19. The validation of a combination of two molecules showing a partial antiviral activity in vivo is therefore of interest. However, this combo does not block viral replication in the nose and is inefficient when the treatment is added after infection, limiting the use of these molecules to prevent people in contact with COVID-19 patient of being infected. However, the authors should demonstrate that their molecules block viral transmission.

    __ Advance:__ The number of antivirals used in the clinics to treat COVID-19 patients remains extremely limited. Increasing the number of drugs available is still sorely needed. Audience: This paper potentially of large interest since the general population has been well informed of and/or have experienced COVID-19. Therefore, it is of interest beyond the virology and infectiology fields.

    Reviewer #3 (Evidence, reproducibility and clarity):

    Summary: In manuscript reference RC-2023-02113, the authors addressed the impact of inhibitors of cell host factors as therapeutics against SARS-CoV-2 infection. They tested the combined inhibition of the enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2, known as essential to meditate viral entry pathways: Conclusion: They showed a reduction, as assessed in vitro experiment (cell line) and in lung infection in mice intranasally- infected with SARS-CoV-2 beta. Moreover, the reduced viral infection is, as expected, associated to lower cell damage.

    Reviewer #3 (Significance (Required)):

    Positive points:

    • The topic is of interest.

    • Robust impact of the treatment although kinetic analysis post infection/symptoms are missing. Limitations:

    • Such a robust level of infection in this model (female BALB/c mice) is surprising, owing that the ACE is not the appropriate homologue. RESPONSE: We respectfully disagree with this concern. The BALB/c strain employed in the current study can be infected by the natural Beta variant, with mutations in the viral spike that allow it to bind to the murine ACE2 receptor and hence can efficiently infect the mice, as we previously described (Kant et al., 2021. Viruses).

    We chose the wt BalB/c model as it better mimics natural respiratory infection in human patients, while the transgene K18-hACE2 model also results in strong infection of the brain. As discussed above, while infection with the Beta variant is efficient, it is not associated with clinical signs, it has only limited pathological effects (mild tissue damage and very limited inflammatory response) and is naturally cleared after 4 days. The ancestral Wuhan strain of SARS-CoV-2 as well as most other variants, in contrast, are unable to bind murine ACE, hence would require the use of transgenic mouse models expressing the human ACE receptor.

    • It would have been interesting to complete the experiments with a demonstration that the compounds block viral transmission. RESPONSE: We apologize for our oversight of not including the statistical analyses in the original version of the manuscript. As requested, it is now included. We are pleased to confirm that in all cases, the differences were statistically significant between presence and absence of combined drugs, and fully support our original conclusions.
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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In manuscript reference RC-2023-02113, the authors addressed the impact of inhibitors of cell host factors as therapeutics against SARS-CoVé infection. They tested the combined inhibition of the enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2, known as essential to meditate viral entry pathways: Conclusion: They showed a reduction, as assessed in vitro experiment (cell line) and in lung infection in mice intranasally- infected with SARS-CoV-2 beta. Moreover, the reduced viral infection is, as expected, associated to level cell damage.

    Positive points:

    • The topic is of interest
    • Robust impact of the treatment although kinetic analysis post infection/symptoms are missing

    Significance

    Positive points:

    • The topic is of interest
    • Robust impact of the treatment although kinetic analysis post infection/symptoms are missing

    Limitation

    • Such a robust levels of infection in this model (female BALB/c mice) is surprising, owing that the ACE is not the appropriate homologue.
    • Statistical analysis are missing for most of the results

    Should be improved to support the strong conclusions.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper, the authors tested the antiviral activity of a combination of compounds by intranasal instillation in a mouse model of SARS-CoV-2. The two compounds used are PIKfyve Kinase inhibitor apilimod dimesylate, which inhibits endosomal maturation, and TMPRSS2 protease inhibitor nafamostat mesylate. The authors have previously shown that a combination of these two inhibitors acts synergistically to prevent entry and infection of SARS-CoV-2 in cell culture. Here, they further investigated the anti-SARS-CoV-2 activity of their combination of compounds by in vivo testing. They used Balb/c mice intranasally inoculated with the Beta variant of SARS-CoV-2. Their data show that concurrent administration of the combo together with the virus prevented lung infection without blocking nasal replication. Delayed administration of the compounds did not reduce replication in the lungs. The only effect was a decrease in bronchiolar cell death. Furthermore, they also tested the stability of the combo at room temperature and their data indicate that these compounds can be kept at room temperature for at least 3 months without losing antiviral activity, at least when resuspended in water. These data are potentially interesting but they need to be consolidated by additional experiments.

    Major comments:

    1. The authors only present immunohistochemistry to investigate viral replication in the nose. A quantitative analysis of replication would allow for better conclusions concerning viral replication in this organ.
    2. Complementary investigation on a potential anti-inflammatory effect of the drugs would also be welcome. Furthermore, it is surprising that the authors did not report potential weight changes.
    3. It would have been interesting to complete the experiments with a demonstration that the compounds block viral transmission.

    Minor comments:

    1. The second paragraph of the introduction is not clear. It needs to be re-written. Furthermore, there is no evidence that Calu3 cells do not express cathepsins.
    2. Figure 4C: Is there any explanation for the lack of apoptosis? The authors should at least provide some hypotheses. Furthermore, this figure is quoted as Figure 4B in the text instead of Figure 4C.
    3. Line 199: The authors claim that the effect of their combo is synergistic. However, this cannot be clearly concluded without appropriate additional experiments where they vary the concentration of the compounds.
    4. Line 211: The sentence is incomplete
    5. The lettering in the panels needs to be doublechecked.

    Significance

    General assessment:

    Finding new antiviral against SARS-CoV-2 remains a priority to fight against COVID-19. The validation of a combination of two molecules showing a partial antiviral activity in vivo is therefore of interest. However, this combo does not block viral replication in the nose and is inefficient when the treatment is added after infection, limiting the use of these molecules to prevent people in contact with COVID-19 patient of being infected. However, the authors should demonstrate that their molecules block viral transmission.

    Advance:

    The number of antivirals used in the clinics to treat COVID-19 patients remains extremely limited. Increasing the number of drugs available is still sorely needed.

    Audience:

    This paper potentially of large interest since the general population has been well informed of and/or have experienced COVID-19. Therefore, it is of interest beyond the virology and infectiology fields.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In this work, Kant and co-workers describe a two drugs regimen for therapeutics treatment of SARS-CoV-2 infection. SARS-CoV-2 infection of cells is dependent on the cleavage of the spike S protein by cellular proteases that prime S allowing the envelop protein to fuse of host membrane during entry and delivery of the viral genome to the target cell. The most important cellular protease is TMPRSS2 located at the surface of the cell. However, in cells with TMPRSS2 levels, Cathepsins, located in endosomes have been shown to be able to also prime S. The therapeutic strategy of the authors relies on the combined usage of an inhibitor of TMPRSS2 (nafamostat) together with a compound that impairs endosomal maturation (apilimod) which is a key step for the activation of cathepsin. The rational is that a dual regimen would be more effective to inhibit SARS-COV-2 infection. Using cell lines and a combination of SARS-CoV2 infection and pseudotyped VSV particles (VSV virus where the glycoprotein has been replaced by the SARS-CoV-2 spike proteins), the authors could show that a two drug regimen was more efficient in preventing SARS-CoV-2 infection compare to single drug regimen. The authors next employed a mouse model of SARS-CoV-2 infection and similarly could show that bi-therapy was more efficient in preventing infection. Importantly, the authors describe a new formulation of the drugs that improve stability of the compounds and shelve life which could be of great benefit with respect to storage needs in therapeutic setting of the population. While the reviewer think the work is potentially very relevant, some of the conclusions are not fully supported by the data and additional experiments/quantifications should be performed to improve rigor and fully support the author conclusions.

    Major comments

    • Throughout the paper, statistical analysis of the results should be performed to support the conclusion of the authors. Currently many experiments do not have statistical analysis and P values or statical significance are missing in most of the figures: Figure 1B, 1D, 4A, 5B,and S2.
    • Quantification of the various pathology observed in mice should be quantified and scored. In the current version, the authors provided a supplementary table describing the pathology observed in individual mice upon SARS-CoV-2 infection. Adapted scoring of the different pathologies should be performed to obtain a statistical view of the pathology induced by SARS-CoV-2 and how this I prevented by the mono and bi-therapy approaches. Additionally, table 1, is very difficult to read as mice are classified in 3 experiments but this does not match with the individual figures, making it very hard to look for the phenotypes. Is it an order issue within the table or are murine infection experiments performed in the order described in table 1?. In this case, can the data be compared between the experiments as some conditions belong to experiment 2 and other to experiment 3? Given the low number of mice, do the experiments have statistical power? To show that treatment of mouse at 3 or 6hpi indeed reduce the number of capsase positive cells, the authors should perform a complete quantification and not limit there analysis to one representative tissue section from one animal
    • the authors insist on the new formulation that improves drug stability. To make this statement, this will need to be actively tested both in cell culture and in animal models. Currently, the authors test the drugs stored 3 months at 25c or -20c and show that they remain active, but in this experiment freshly made drug was not directly tested in parallel. Additionally, to make such a statement, different concentration of the drugs should be tested to calculate a IC50 for freshly prepared drug and stored drugs (as the current concentration tested might be at saturating concentration). Finally, the mouse experiments are performed with freshly made compounds and if the authors want to highlight the new formulation and increased stability, experiments in mice should be performed also with stored compounds. Alternatively, statement on drug stability should be removed or strongly tuned down from text.
    • Statistical analysis on figure 2b should be done between nafamostat alone and dual treatment to show that both drugs are cooperative in term of antiviral activities
    • The authors state "A quantitative assessment of the in vivo synergy is shown here by the enhanced decrease of viral RNA in lungs of mice treated with both drugs at very low concentrations (Figure 2 B, compare using 2 mg/Kg apilimod dimesylate and 4 mg/Kg nafamostat mesylate alone, and in combination)." I guess, the authors want to comment on the fact that 0.2 mg/kg of apilimod and 0.4 mg/kg of nafamostat are as potent as 2 and 4 mg/kg. is that correct? If YES, to make this statement, bi-therapy should be compared to mono-therapy at the same concentration.
    • when drugs are injected after infection (Fig 4), the drugs are not active. In fact, unless the reviewer mis-understood the plot, the mouse are even more infected compared to vehicle. The authors wrote that both regimes are equally less effective compared to drug administered during infection. The authors should write that both regimes are equally none protective. If drugs are not active after infection, does this approach really represent a therapeutic solution. The authors suggest that it does by limiting pathologies but this needs to be better quantified (see comment above)
    • In the rebound experiment: unless the reviewer misunderstood, it appears that no conclusion can be driven from this experiment. Q-PCR data for vehicle animal a 4dpi show no sign of infection, so the experiment is not really interpretable since control animals are no longer positive. The authors suggest that there is less pathologies but this needs to be better quantified (see comment above)

    Minor comments

    • It will make reading easier if the authors always mentioned which drugs inhibit what. For example: addition of the TMPRSS2 inhibitor nafamostat etc.... or addition of apilimod to block cathepsins activities.....
    • Figure 1: make a comment in the text that cells with low TMPRSS2 are more sensitive to the cathepsin inhibitor apilimod and vice versa, cells with high TMPRSS2 are more sensitive to nafamostat. This is expected and it could be highlighted.
    • Figure 2B: how are the data normalized?. should not RdRp, E and SubE all have a mean at 100% for the vehicle?
    • Line 211: something is missing here "when (Fig 2...)
    • Line 221 should figure 4c
    • Figure legends should only contain the details of the experimental design but should not contain description and interpretation of data. This is very minor and maybe a question of taste.

    Referees cross-commenting

    the other reviewers have highlighted the same limitations concerning the lack of quantifications of the immunochemistry and also the lack of robust statistical analyses.

    this should be highlighted to the authors as it appears to be the minimum to do prior publication. this should not take too much time as the data are in principle already available

    Significance

    The work by Kant and co-workers is potentially very significant but some limitations (as highlighted above) impair the impact of the work in his current version. The approach employing a two-drug regimen to combat SARS-COV-2 infection by targeting both TMPRSS2 and cathepsin activities is not new and was described before by the authors themselves. Employing this approach in an animal model is new and the new formulation improving drug stability and facilitating storage could be a game changer in therapeutic setting of patients. As such, this work could be highly significant and of broad interest. However, additional experiments and clarifications are needs to elevate this work to high impact standards. The reviewer believes that the requested experiments are easily achievable by the research teams of this project and think that the project will ultimately have a strong impact in the field.

  5. Version published to 10.1101/2023.07.19.549731v1 on bioRxiv