A versatile Plasmodium falciparum reporter line expressing NanoLuc enables highly sensitive multi-stage drug assays

Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial drugs effective against multiple stages of the parasite, we generate a P. falciparum reporter parasite that constitutively expresses NanoLuciferase (NanoLuc) throughout its whole life cycle. The NanoLuc-expressing P. falciparum reporter parasite shows a quantitative NanoLuc signal in the asexual blood, gametocyte, mosquito, and liver stages. We also establish assay systems to evaluate the anti-malarial activity of compounds at the asexual blood, gametocyte, and liver stages, and then determine the 50% inhibitory concentration (IC50) value of several anti-malarial compounds. Through the development of this robust high-throughput screening system, we identify an anti-malarial compound that kills the asexual blood stage parasites. Our study highlights the utility of the NanoLuc reporter line, which may advance anti-malarial drug development through the improved screening of compounds targeting the human malarial parasite at multiple stages.

of ATQ was evaluated with a higher sensitivity than the already published parasite line of reference 14. However, the IC50 values obtained in both systems are within the same order of magnitude and there is no way to directly correlate one to the other.
Lines 408-410 -It is said that "Exp227 was used for the NanoLuc reaction at oocyst and sporozoite stages". Has this line been characterized? This data should be included as supplementary results.
The authors could consider briefly mentioning in the discussion other, non-human, reporter Plasmodium parasite lines expressing luciferase that allow, for example, to test the activity of compounds in vivo, in rodent malaria models. In this line of thought, although not mandatory for publication, demonstrating that the GFP-NanoLuc reporter parasite line could be used to measure infection through bioluminescence in the liver or blood of liver-or blood-humanized mice would certainly be of added value to the manuscript. Figure 1D -it is not clear in which sample this expression is being analysed. Please clarify. Figure 2D -"Dose-response curves of established antimalarial compounds" would be a more suitable description of the plots, instead of "Determination of IC50 value", given that this is only shown in the table. Inhibition is represented as a % of the control, however, it is not clear what that control is. Figure 3D -the control to which inhibition is being normalized to should be described in the figure legend.   figure 5E shows the impact of ATQ on the percentage of infected hepatocytes, which is not a measure of intra-hepatic parasite development. Have the authors assessed EEF areas? RLU measurements will not allow for the discrimination between the impact of a drug on hepatocyte invasion and intra-hepatic development. A direct comparison between the data of Fig 5D and Fig 5E should not be made without discussing these points. Figure 6 -Inhibition is represented as a % of the control, however, it is not clear what that control is. In Fig 6G, the axis is misleading and it does not immediately convey the idea that the readout is cytotoxicity. Table 1 -the SD of stage III gametocytemia for GFP-NanoLuc should be within brackets for consistency. The meaning of (4 exp.), etc, should be clarified. Given that stage III gametocytemia was not determined for Pf NF54 WT but is referred to in the text as being normal, there should be a literature reference to support this conclusion.  Figure S1 -there is a contradiction between the expected amplicon size shown in S1A resulting from the use of primers p1 and p5 (1.5 kb) and that shown in S1B and described in the figure legend (1.1 kb). This should be clarified. Do the authors have an explanation for the presence of a band of ~1.5 kb in the first gel lane?

TABLES, FIGURES, AND FIGURE LEGENDS
For all correlation graphs, it is said in the figure legend that the solid line indicates the mean RLU from triplicate samples. Isn't that instead indicated by the dots and doesn't the line correspond to the correlation itself?
Reviewer #2 (Remarks to the Author): The study by Miyazaki et al demonstrates the usefulness of transgenic parasites for drug development in malaria. The transgenic reporter line that was produced in this study overcomes several limitations with previously generated reporter lines. These include that; 1) it is a dual reporter for fluorescent read out and luciferase-based readout, 2) the nanoluciferase used is significantly brighter than other luciferases that have been used, which is a significant advantage when using low cell numbers such as for gametocytes, mosquito stages and liver stages, and 3) the reporter is introduced into the genome in a stable and marker free method making it suitable for all parasite lifecycle stages. Furthermore, the authors made improvements to assay format that overcome limitations with leftover signal from dead parasites in the gametocyte assay. Using these methods, the authors demonstrated that the reporter line is suitable for drug screening throughout the lifecycle stages. As a proof of concept, the authors performed a HTS screen of 1920 compounds against asexual stages, and 30 hits from the screen against gametocyte stages. This lead to the finding of OU0074008 with activity against both stages that could be a promising lead compound for further development. However, several issues do need to be addressed. The main issue is the lack of clear reporting of the number and type of replicates used in each figure (see below).
Major comments: 1. In the majority of figures there is insufficient detail on the number of replicates shown. E.g. 2B and 2C have no information about replicates. For figure 5, it states the mean of quadruplicates. It is unclear if these are technical duplicates from a single experiment, or biological quadruplicates, in which case was it a single technical replicate in each condition? These comments apply to the majority of figures in this study. 2. Lines 241-242. A common problem with luciferase as the readout for assays is the ability of small molecules to directly inhibit luciferase activity. Given the significant difference between the asexual and gametocyte assay, it is very likely it is specific against parage growth in asexuals. However, it may be that the gametocyte activity is due to luciferase inhibition. Either of the following experiments would clarify this. 1) A counter screen for luciferase inhibition. Directly test for inhibition of luciferase using lysate from asexual parasites (lysing them and adding drug -at the same concentrations used in the gametocyte assay -directly to the lysate for a short period prior to adding substrate). 2) repeat the gametocyte assay using an alternative read out for the assay.
Minor comments 3. Figure 2B, how was the serial dilution performed? If it was diluted into the equivalent amount of blood lysate the signal at low parasitemia becomes quenched more so (due to hemoglobin) than if the sample was diluted in PBS or equivalent (Azevedo 2014). 4. A comment on the advantages and limitations of the gametocyte assay would be useful. For example, do you know if your assay is suitable for separating out early vs late stage gametocyte activity (e.g. Duffy et al, 2013, Malaria Journal). 5. Figure 4D. It is very difficult to see the individual data points at the low end. A log scale, or a split axis would help this. 6. Figure 5C, the labels are positioned in such a way that they look like two data points in D7.
Reviewer #3 (Remarks to the Author): bright Nanoluc luciferase and the fluorescent GFP (or mCherry). The line does not contain a selectable marker and shows reporter expression throughout the life cycle. The others nicely show that that the constitutively expressed reporters do not appear to have any effect on the growth characteristics of the parasites at the different developmental stages of the life cycle. They propose that this line is suitable for the multi-stage evaluation of anti-malarial drug efficacy and using this line in HTS against asexual blood stage parasites and gametocytes, they identify OU0074008 as a novel anti-malarial compound.
While many different P. falciparum reporter lines have been developed, this new reporter line is attractive, because it is marker-less, has constitutive expression of GFP for visualization/isolation and the bright Nanoluc for bioluminescence read-out for drug assays at multiple stages of the parasite life cycle and appears to have a normal viability. This 'one-for-all' line could simplify screening of compounds directed against different parts of the life cycle of P. falciparum.
This manuscript represents a nice piece of work with investigations covering the full life cycle of the parasite. In my opinion however, some of the claims, especially claims regarding the performance of the parasite line during the liver stage part of the life cycle are not fully substantiated (see below).
Major comments 1. The analyses of the liver stage parasites by bioluminescence measurements appear to be limited. In the abstract (line 33) 'a quantitative' NanoLuc signal in liver stages (amongst other stages) is mentioned and it is stated that an assay system was established for asexual, gametocyte, and liver stages (line 35). In line 276 it is stated that luminescence and parasite number are well correlated, but this is not (clearly) shown for liver stages. And in the conclusion (lines 322 and 323) it is stated that a robust drug assay protocol is established for asexual-, gametocyte-, and liver-stage parasites. For asexual stages and gametocytes, relationship between parasite number and RLU, drug assay parameters (Z' factors etc.) have been described and multiple drugs were tested. However, for liver stage parasites, this is not the case. Only data of a single (?) drug experiment with atovaquone are shown. To substantiate the claim that a robust liver stage drug assay based on bioluminescence with these parasites has been developed, more experimentation is needed. Similar to the work performed on the asexual-and gametocyte stages, the relationship between RLU values and parasite numbers needs to be assessed and multiple drug assays to determine activity with different drugs should be performed for liver stages as well.
2. Also, the claim that the activity of atovaquone against liver stage P. falciparum can be evaluated with a 'higher sensitivity' (line 301) compared to a previously established similar system by Marin-Mogollon et al (ref 14) needs more extensive experimentation (in fact IC50 values from both methods appear to be similar).
3. In the light of using a single reporter line for multiple stages of the life cycle, it would make sense to test the novel identified anti-malarial compound OU0074008 for asexual and gametocyte stages also in the liver stage assay.
Minor points 1. The use of a single reporter line for the investigation of multiple stages of the life cycle would simplify the drug screening work. However, aren't the authors concerned that cross-contamination of stages (i.e. presence of remaining blood stages in the gametocyte preparation) could give problems in assessing drug activity against specific stages? 2. For the figures with IC50 curves, the following details should be clarified and mentioned: how many times was the assay performed (independently); how many wells were used per assay (96 or 384 well format); what is depicted, for example triplicates of wells or triplicates of assays? 4. The rationale for using NanoLuc is not completely clear from the introduction (also, please provide a ref for NanoLuc in line 87).
5. Line 112/113: was the in vitro growth of the mCherry-NanoLuc line also comparable to the WT strain?
6. Lines 166, 167: wouldn't these prolonged incubation times affect parasite viability? Did the authors check whether these gametocytes could still be transmitted? And what is the transmission rate of the gametocytes after the different incubation times? 7. Line 179: could the need for the NanoLuc inhibitor indicate prolonged stability of the protein? If so, do the authors think that it would be an option to develop a NanoLuc reporter with a degradation domain to counteract this issue? 8. Lines 225, 226: were all HSP70 parasites also GFP positive (indication of the stability of the transgenes in the genome)? 9. Figure 5: panel E: on the y-axis % infected hepatocytes is stated. How was this calculated? What is the number of liver stages per well? Is this a single assay from which means and SD of triplicate wells are shown? I would strongly suggest using a more commonly used parameter, such as % of untreated control instead.
10. Lines 299-302: the authors state that the liver stages could be measured with high sensitivity. I do think that more data are needed for this statement (see above). Also, the RLU values appear to be low (around 4000) if these reflect parasite numbers around 50-100 per well. In papers describing Nanoluc in liver stage parasites from other malaria parasites (

Response letter for the manuscript entitled "A versatile Plasmodium falciparum reporter line expressing NanoLuc enables highly sensitive multi-stage drug assays"
Our responses are indicated in a blue font.

Reviewers' comments:
We sincerely thank the Reviewers and Editors for providing their valuable comments and suggestions to improve our manuscript. In response to the issues raised by the reviewers, we performed several new experiments and thoroughly revised the manuscript to address all the comments raised. We believe that our new data and revisions have greatly improved the quality of the manuscript, thereby making it suitable for publication.

Reviewer #1 (Remarks to the Author):
Miyazaki Y. and colleagues have generated a Plasmodium falciparum reporter line that expresses GFP and NanoLuc. NanoLuc expression has been confirmed and employed in the evaluation of the anti-plasmodial activity of compounds against asexual blood, gametocyte and liver parasite stages. Employment of this transgenic parasite for the screening of a library of 1920 compounds led to the identification of a hit compound that is active against both asexual blood and gametocyte P. falciparum stages. Even though other reporter parasite lines that express luciferase (firefly or NanoLuc) have been described, this is the first P. falciparum line expressing NanoLuc that has been validated for the evaluation of anti-plasmodial activity against gametocyte and liver stage parasites, besides asexual blood stage parasites. The greater brightness of the NanoLuc signal, when compared to firefly luciferase, increases the sensitiveness of parasite detection. Furthermore, the confirmation of its expression across the parasite's life cycle makes it a useful tool in the search for compounds with multistage activity. Overall, the paper is clearly written and the claims are supported by the evidence presented. However, I have some minor concerns that the authors should address before publication of this manuscript.
We would like to thank the reviewer for the positive comments and informative suggestions on how to improve the manuscript.

MANUSCRIPT TEXT
Line 48 -"Parasites in the hepatocytes eventually lead to rupture". Intra-hepatic parasites are released from hepatocytes in merosomes, not upon hepatocyte rupture. The sentence should be rephrased accordingly.
As per the reviewer's suggestion, we have revised the sentence as follows: Line 48-50 "Parasites in the hepatocytes are eventually released into the bloodstream, and subsequently invade RBCs for further proliferation." For clarity, "asexual stages" could instead be referred to as "asexual blood stages" across the manuscript text.
We have revised all the instances of "asexual stages" to "asexual blood stages" in the manuscript, as suggested.
Line 131 -"For subsequent analysis, we mainly used the GFP-NanoLuc reporter line". This sentence is too vague, and the authors should clarify when and why one or the other parasite line was used. The mCherry reporter line is not clonal. If this was a reason for not using it, this should be stated. Line 185 -please consider revising the statement as it appears that the results of this section support that the parasite line is suitable for asexual blood stage assays, a claim that in fact is supported by the findings of the previous results section.
As suggested, we have revised this sentence to clarify the meaning of this paragraph. We have also deleted "as well as for assays performed at the asexual stage." from the original sentence .
"These findings demonstrated that the GFP-NanoLuc line is applicable for P. falciparum In this study, we screened 1920 compounds using asexual blood stages and selected only 30 compounds for the downstream gametocytocidal assay. As shown in Table 3, the 96-well plate gametocytocidal assay is a more robust drug assay system than the 384-well format, although the throughput of the 96-well format is lower. To screen only 30 compounds, we considered the 96well formats as a suitable assay system.
We have revised the sentence in Lines 255-258 to address this issue as follows "For the gametocytocidal assays, we applied the 72 h+0 h protocol with the extracellular NanoLuc inhibitor in a 96-well plate format considering that it exerted high assay quality and required a short time (Table 3)." Lines 243-245 and Figure 6G: The IC50 value against HepG2 cells is said to be higher than that against the asexual blood stage parasite. However, this value is not presented and the data does not appear to allow for an IC50 determination. The axis labeling is also misleading, as it does not clearly indicate that what is being measured is toxicity towards the human hepatic cell line at the compound concentrations tested. This is confusing and should be clarified.
We agree with the reviewer's comment. To address this, we repeated the cytotoxicity assay by applying a more dynamic range of the compound, which determined the IC50 value of OU0074008 against HepG2 cells. This new data has been added to Fig. 6G by changing the Yaxis to cytotoxicity (% of control), as suggested by the reviewer. The new Fig.6 is shown below.
Line 299 -This sentence could be rephrased for clarity. As it is written, it appears that the activity of ATQ was evaluated with a higher sensitivity than the already published parasite line of reference 14. However, the IC50 values obtained in both systems are within the same order of magnitude and there is no way to directly correlate one to the other.
We agree with this suggestion. We have rephrased this sentence as follows (Line 330-334): 'Moreover, we demonstrated that the activity of atovaquone and MMV390048 against liver stage P. falciparum in primary human hepatocytes can be evaluated using our drug assay system with brighter luminescence when compared to a previously established system using Firefly luciferase.
Lines 408-410 -It is said that "Exp227 was used for the NanoLuc reaction at oocyst and  Figure S3 is shown below.
The authors could consider briefly mentioning in the discussion other, non-human, reporter Plasmodium parasite lines expressing luciferase that allow, for example, to test the activity of compounds in vivo, in rodent malaria models. In this line of thought, although not mandatory for publication, demonstrating that the GFP-NanoLuc reporter parasite line could be used to measure infection through bioluminescence in the liver or blood of liver-or blood-humanized mice would certainly be of added value to the manuscript.   This issue is the same as the one mentioned above. The legend for Fig.3 has been revised as according to the above-mentioned modification (Line 766-769).   is. In Fig 6G, the axis is misleading and it does not immediately convey the idea that the readout is cytotoxicity.
This issue is the same as that described above. The legend for Figure.6G has been revised as according to the above-mentioned modification (Line 834-836).
"The cells were exposed for 72 h with indicated concentration of OU0074008 or DMSO (control well) on a 96 well plate and cytotoxicity assay using CCK-8 was performed." Table 1 -the SD of stage III gametocytemia for GFP-NanoLuc should be within brackets for consistency. The meaning of (4 exp.), etc, should be clarified. Given that stage III gametocytemia was not determined for Pf NF54 WT but is referred to in the text as being normal, there should be a literature reference to support this conclusion.  We have revised Table 2 according to your suggestion (Line 847-848). Figure S1 -there is a contradiction between the expected amplicon size shown in S1A resulting from the use of primers p1 and p5 (1.5 kb) and that shown in S1B and described in the figure   legend (1.1 kb). This should be clarified. Do the authors have an explanation for the presence of a band of ~1.5 kb in the first gel lane?
Thank you for spotting this error, which shows that the amplicon by 5'-int primers was 1.5 kb.
We have revised this information based on your suggestion. We rarely amplify a ~1.5 kb band in WT by primer P1/P5, as we previously reported ( The study by Miyazaki et al demonstrates the usefulness of transgenic parasites for drug development in malaria. The transgenic reporter line that was produced in this study overcomes several limitations with previously generated reporter lines. These include that; 1) it is a dual reporter for fluorescent read out and luciferase-based readout, 2) the nanoluciferase used is significantly brighter than other luciferases that have been used, which is a significant advantage when using low cell numbers such as for gametocytes, mosquito stages and liver stages, and 3) the reporter is introduced into the genome in a stable and marker free method making it suitable for all parasite lifecycle stages. Furthermore, the authors made improvements to assay format that overcome limitations with leftover signal from dead parasites in the gametocyte assay. Using these methods, the authors demonstrated that the reporter line is suitable for drug screening throughout the lifecycle stages. As a proof of concept, the authors performed a HTS screen of 1920 compounds against asexual stages, and 30 hits from the screen against gametocyte stages.
This lead to the finding of OU0074008 with activity against both stages that could be a promising lead compound for further development. However, several issues do need to be addressed. The main issue is the lack of clear reporting of the number and type of replicates used in each figure (see below).
We would like to thank the reviewer for the useful suggestions for enhancing the quality of our manuscript that we have incorporated in the updated manuscript. 2. Lines 241-242. A common problem with luciferase as the readout for assays is the ability of small molecules to directly inhibit luciferase activity. Given the significant difference between the asexual and gametocyte assay, it is very likely it is specific against parage growth in asexuals.
However, it may be that the gametocyte activity is due to luciferase inhibition. Either of the following experiments would clarify this. 1) A counter screen for luciferase inhibition. Directly test for inhibition of luciferase using lysate from asexual parasites (lysing them and adding drug -at the same concentrations used in the gametocyte assay -directly to the lysate for a short period prior to adding substrate). 2) repeat the gametocyte assay using an alternative read out for the assay.
We agree with this suggestion. To address these issues, we performed new experiments regarding the direct inhibition of NanoLuc by OU0074008 using a lysate of asexual blood and gametocyte stages (Counter assay). In addition to the NanoLuc inhibition assay, we performed an LDH assay using asexual blood stages treated with OU0074008 to further verify the antimalarial activity of "To verify the anti-malarial activity of OU0074008, we performed a lactate dehydrogenase (LDH) assay and confirmed that the IC50 value from different types of viability assay was equivalent to that from the NanoLuc assay (Fig.S5). Furthermore, the NanoLuc inhibition assay with OU0074008 suggests that there is no obvious NanoLuc inhibitory activity of the hit compound ( Fig.S5)." (Line 263-268).
Minor comments 3. Figure 2B, how was the serial dilution performed? If it was diluted into the equivalent amount of blood lysate the signal at low parasitemia becomes quenched more so (due to hemoglobin) than if the sample was diluted in PBS or equivalent (Azevedo 2014).
As described in the Methods section (Line 540-542), we performed a serial dilution with a medium containing uninfected RBCs at the same hematocrit (2%) as that of the culture to normalize the background. In our case, as shown in Fig. 2B, the correlation was linear, at least between 2 x 10 5 -1 x 10 2 parasites and no effect on the signal was observed, even at low parasitemia.

Advantages:
The gametocytocidal assay with the GFP-NanoLuc line is a simple and user-friendly system that enables high-throughput screening with high sensitivity in any laboratory as long as standard equipment is prepared. Gametocyte purification with a magnet or percoll to remove the effect of background derived from uninfected RBCs and IFA to visualize parasites, both of which lower the throughput significantly, is not necessary for our assay system. In addition, expensive imaging devices to quantify parasite numbers, such as a high-content imaging system, are not necessary. Additionally, it is theoretically possible to investigate the antimalarial effect of compounds at the early gametocyte stage, which is achieved by shortening the culture time post-gametocyte induction, although we did not perform an assay with early-stage gametocytes in this study. In this case, the purification of stage I gametocytes may be required before seeding the parasites on an assay plate.

Limitations:
The only limitation of our protocol is that the costs for NanoLuc inhibitors provided by Promega are relatively high, which is not suitable for high-throughput screening with limited resources. 5. Figure 4D. It is very difficult to see the individual data points at the low end. A log scale, or a split axis would help this.
As suggested, we have revised Fig.4D and Fig.4E to clearly show the value at the low end (Line 773). New Fig.4D and 4E are shown below.
6. Figure 5C, the labels are positioned in such a way that they look like two data points in D7.
As suggested, we have revised Fig.5C (Line 792-793). New Fig.5C  We would like to thank the reviewer for the informative suggestions for improving our manuscript that we have incorporated in the updated manuscript.
Major comments 1. The analyses of the liver stage parasites by bioluminescence measurements appear to be limited.
In the abstract (line 33) 'a quantitative' NanoLuc signal in liver stages (amongst other stages) is mentioned and it is stated that an assay system was established for asexual, gametocyte, and liver stages (line 35). In line 276 it is stated that luminescence and parasite number are well correlated, but this is not (clearly) shown for liver stages. And in the conclusion (lines 322 and 323) it is stated that a robust drug assay protocol is established for asexual-, gametocyte-, and liver-stage parasites. For asexual stages and gametocytes, relationship between parasite number and RLU, drug assay parameters (Z' factors etc.) have been described and multiple drugs were tested.
However, for liver stage parasites, this is not the case. Only data of a single (?) drug experiment with atovaquone are shown. To substantiate the claim that a robust liver stage drug assay based on bioluminescence with these parasites has been developed, more experimentation is needed. Similar to the work performed on the asexual-and gametocyte stages, the relationship between RLU values and parasite numbers needs to be assessed and multiple drug assays to determine activity with different drugs should be performed for liver stages as well.
In the revised manuscript we now include an analysis of parasite numbers versus luciferase signals at day 4, 5 and 6 post infection in Figure 5 (Line 792-793). In addition, we include assay parameters for the liver stage assay in Table 3  We agree with this comment and have revised the corresponding sentence accordingly. We did not validate that the liver-stage assay established in this study has a higher sensitivity when compared to a similar assay system that Marin-Mogollon et. al. previously established. As this sentence is misleading, we have rephrased it as follows (Line 330-334).
'Moreover, we demonstrated that the activity of atovaquone against liver stage P. falciparum in primary human hepatocytes can be evaluated using our drug assay system with brighter luminescence when compared to a previously established system using Firefly luciferase.' 3. In the light of using a single reporter line for multiple stages of the life cycle, it would make sense to test the novel identified anti-malarial compound OU0074008 for asexual and gametocyte stages also in the liver stage assay.
We agree with this suggestion. In our initial trials, we tested OU0074008 with liver-stage parasites of another P. falciparum reporter line (not the GFP-NanoLuc line generated in this study) due to limitation in our laboratory, and we found that the compound has no anti-malarial effect on the liver-stage parasites. This data is included in Fig.S6 (Line 268-270, 957) and shown below. Thus, we considered that another compound known to be effective on liver-stage P. falciparum parasites is preferable for validating the liver-stage assay using the GFP-NanoLuc line than OU0074008. Minor points 1. The use of a single reporter line for the investigation of multiple stages of the life cycle would simplify the drug screening work. However, aren't the authors concerned that crosscontamination of stages (i.e. presence of remaining blood stages in the gametocyte preparation) could give problems in assessing drug activity against specific stages?
We believe that each assay established in this study showed stage-specific results. During gametocyte preparation, the asexual blood-stage parasites were killed by N-acetylglucosamine (NAG), as evidenced by the lack of antimalarial effects of atovaquone in the gametocyte-stage assay. If asexual stage parasites are left in the preparation, the luminescence intensity of the DMSO well should increase after 72 h of incubation, and there will be a difference in the signal intensity between the DMSO and the atovaquone wells.
2. For the figures with IC50 curves, the following details should be clarified and mentioned: how many times was the assay performed (independently); how many wells were used per assay (96 or 384 well format); what is depicted, for example triplicates of wells or triplicates of assays?
We have revised this according to the suggestion by reviewer 2, to include biological and technical replicates in the Figure  As per your suggestion, we have added new sentences in the introduction section (Line 88-91).
"NanoLuc is a suitable reporter protein for high throughput screening, which has a higher luminescence intensity (80-to 240-fold) and structural stability than firefly luciferase (Hall, M.P., et al. (2012) ACS Chem Biol 7:1848)." 5. Line 112/113: was the in vitro growth of the mCherry-NanoLuc line also comparable to the WT strain?
As described in Fig.S1B, the mCherry-NanoLuc reporter line is a mixture of integrated parasites and NF54 WT. Hence, we did not examine the in vitro growth of the mCherry-NanoLuc line, although this line should show normal growth in the asexual blood stages.
6. Lines 166, 167: wouldn't these prolonged incubation times affect parasite viability? Did the authors check whether these gametocytes could still be transmitted? And what is the transmission rate of the gametocytes after the different incubation times?
We did not check the gametocyte viability and transmissibility after incubation with compounds because this is beyond the focus of the manuscript. We established a multi-stage drug assay including the asexual blood, gametocytes, and liver stages. However, since we are also interested in this point, we will consider it in the future. 7. Line 179: could the need for the NanoLuc inhibitor indicate prolonged stability of the protein?
If so, do the authors think that it would be an option to develop a NanoLuc reporter with a degradation domain to counteract this issue?
Yes, we have considered using a NanoLuc reporter with a degradation domain (PEST) and generated a P. falciparum line expressing NanoLuc-PEST. However, contrary to our expectation, the NanoLuc protein derived from killed gametocytes accumulated due to the loss of proteasome degradation activity in the dead cells, thereby resulting in a higher luminescence signal in the positive control (epoxomicin) wells when compared to the DMSO wells. Therefore, we believe that the drug assay using gametocytes of the GFP-NanoLuc line with the extracellular NanoLuc inhibitor described in this manuscript is more robust and suitable for gametocytocidal assays. Double staining for GFP and HSP70 was not performed because the experiment was aimed to examine reporter expression. Since the GFP-NanoLuc line used in the liver stage experiment is clonal, all parasite population is likely to express reporter proteins. 9. Figure 5: panel E: on the y-axis % infected hepatocytes is stated. How was this calculated?
What is the number of liver stages per well? Is this a single assay from which means and SD of triplicate wells are shown? I would strongly suggest using a more commonly used parameter, such as % of untreated control instead.
In light of this comment and the comments raised by reviewer 2, we have adapted Figure 5G to show the absolute numbers of parasites (See above). In addition, we have included a panel 5F that shows the effect of atovaquone on the total parasite area (See above). These experiments show data from three independent replicates, and we have clarified this in the As we replied to the comment raised in 'major point 2,' since this sentence is misleading, we have rephrased it as follows (Line 330-334): "Moreover, we demonstrated that the activity of atovaquone against liver stage P. falciparum in primary human hepatocytes can be evaluated using our drug assay system with brighter luminescence when compared to a previously established system using Firefly luciferase." Regarding the RLU value, thank you for pointing it out. Absolute values of RLU are incomparable between our study and other studies because it varies depending on the detection system and measurement settings (dynamic range) of luminometers used. In addition, since the promoter and the Plasmodium species used in each study differ from those of our GFP-NanoLuc line, it becomes even more challenging to compare the RLU values. Even if the signal intensity is high, it may be because the background signal is high. Relative values of RLU (i.e. Signal/Background: S/B) are more important when comparing the assay quality. We consider that the liver-stage assay using the GFP-NanoLuc line has very high quality since the S/B is high enough (Table 3). 11. Figure 2,  Thank you for spotting this. We carefully checked our original data and noticed that the background signal in the asexual blood stage was much lower than 10 3 . We revised Figure 2B according to the original data. New Fig.2B is shown below.