Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity

  1. Selma Belhimeur
  2. Sylvie Briquet
  3. Roger Peronet
  4. Jennifer Pham
  5. Pierre-Henri Commere
  6. Pauline Formaglio
  7. Rogerio Amino
  8. Artur Scherf
  9. Olivier Silvie
  10. Salaheddine Mecheri

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Abstract

Plasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites.

Methods

Considering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development.

Results and Discussion

Though IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo , these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8 + T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.

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  1. Version published to 10.3389/fimmu.2023.1143012
  2. Version published to 10.1101/2021.11.16.468835v2 on bioRxiv
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    Reply to the reviewers

    Manuscript number: RC-2021-01158

    Doi preprint: https://doi.org/10.1101/2021.11.16.468835

    Corresponding author(s): Salah, MECHERI

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

    __Whole sporozoite vaccines confer sterilizing protection against Plasmodium infection. However, further improvements of whole sporozoite vaccines is needed and requires a thorough understanding of the immune processes that mediate protection and the deployment of novel strategies further augment protective immunity while limiting the impact of factors that are detrimental to protection. Work from the Mecheri laboratory and others had previously established that IL-6 signaling plays a critical role in the immune response to a liver stage infection; engagement of IL-6 signaling promotes the initial control of a liver stage infection and enhances the protective adaptive immune response. Given this potent protective role for IL-6, Belhimeur and colleagues design a parasite strain in rodent malaria parasites that encodes and secrete murine IL-6 during liver stage infection. They show that upon infection of wildtype mice, these transgenic parasites i) are unable to transition to blood stage infection, ii) produce Il-6 and iii) induce a durable adaptive immune response that can protect against sporozoite challenge. This study is novel and intriguing. However, a superficial analysis of the transgenic parasite strain, an incomplete analysis of the immune response to infection and the lack of data regarding the possibility of IL-6 mediated immunopathology have dampened this reviewer's enthusiasm for the work.

    **Major Concerns:** __

    1)The data in Figure 3b-3d clearly indicate that the IL-6 encoding transgenic parasites exhibit a defect in parasite development within HepG2 cells that is maintained in vivo. The authors propose that an arrest of these parasites in the liver stage precludes their transition to blood stage infection and that this arrest is dependent on IL-6 signaling. To better support that claim the authors should:

    a.Better characterize in vivo liver stage arrest using infected liver tissue analysis with immunofluorescence microscopy to determine when and how precisely IL-6 transgenic parasites are impacted in development.

    Done. New data in figure 3B, C, D

    b.Determine if arrested development of IL-6 transgenic parasites is truly dependent on IL-6 signaling using antibody blockade of IL-6 signaling and mice with genetic defects in IL-6 signaling.

    __Experiments were done using anti-IL-6 receptor blocking antibodies, but did not work. This was commented in the text and shown in Supplementary Fig 2 . __

    __ 2)The authors claim that IL-6 production and secretion into the liver tissue augments the adaptive immune response to liver stage infection. This in turn results in a durable adaptive immune responses that protect against infection. However, the mechanistic underpinning of IL-6 signaling in the liver that is induced by their transgenic parasites and the impact on adaptive immune responses is poorly characterized: __

    __a.There is no evidence that the protective adaptive immune response induced by IL-6 trangenic parasite infection is dependent on IL-6 signaling. Is superior protection and immunogenicity lost in IL-6 signaling deficient animals that are infected with IL-6 transgenic parasites? __

    Not addressed but the point is that IL-6 leads to attenuation.

    b.What elements of the adaptive immune response are impacted? One can imagine that IL-6 mediated killing of infected hepatocytes might introduce more parasite antigen that can be acquired by antigen presenting cells, or that IL-6 mediated pro-inflammatory signaling might regulate the maturation of antigen presenting cells, increased differentiation of helper T cells, the downregulation of regulatory T cell function and frequency and/or the differentiation of effector CD8 T cells into long-lived hepatic memory CD8 T cells. The authors should conduct a more comprehensive analysis of how parasite-encoded IL-6 impacts adaptive immunity.

    Done. An extensive analysis of CD4 and CD8 phenotype and status of activation is represented in Fig 9.

    3)While IL-6 transgenic parasites induce a potent and durable adaptive immune response, the authors should show how this compares to published whole sporozoite immunizations. The authors should determine if immunization with IL-6 transgenic parasites is superior to for example immunization with radiation-attenuated sporozoites and generically attenuated sporozoites.

    It not the point. The work presented here emphasizes the proof of concept that the proposed new strategy works. Follow up studies will compare this model to previous ones.

    4) IL-6 signaling is a major player in inflammatory diseases and the induction of immunopathology. As such the authors should carefully examine the duration and magnitude of IL-6 protein production in the liver, and serum after IL-6 Tg parasite infection and determine if IL-6 signaling promotes liver immunopathology.

    Not done but this point was discussed in the text. Also, we made it clear in the material and methods section that the way the construct was made, i.e the IL-6 production is time-frame restricted to the first 48h of liver infection, precisely because of the expression of IL-6 gene is under the control of LISP-2 promoter. Therefore there is no persistence of IL-6 production by liver stage parasites.

    Reviewer #1 (Significance (Required)):

    __ The paper is reporting a novel strategy to generate a whole sporozoite vaccine. Expression of IL6 in a transgenic parasite. This could be a significant contribution to the field if additional experiments as outlined in the critique are conducted.The work might also inform vaccine design for other pathogens. __

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    __The manuscript describes the construction of a Plasmodium berghei that expresses murine interleukin-6 in exoerythrocytic (liver stage) parasites and the analysis of mice infected with sporozoites of this parasite line. They find that such parasites do not complete development in liver cells and therefore do not produce subsequent infection in red blood cells. The ability of prior infection with these parasites on the ability of the host to resist both wild type and heterologous species challenge is then examined. __

    __ The key assumption that underlies the study is that the observed phenotypes result from parasite expression of bioactive IL-6 that functions to modulate the immune system. Other explanations are not considered, for example the over-expression of secreted IL-6 may prevent the complete maturation of the intracellular parasite by clogging up the parasite secretory pathway. The authors use the 'wild type' parasite as the control but not only does the wild type not express IL-6 it also does not express the human DHFR gene used as a selection system. A much better control parasite would be one that expresses a non-bioactive IL-6 so that the potential effects on parasite maturation can be differentiated from those on the mouse immune system. Another control to be considered would be comparison with a genetically attenuated parasite with a block in late stage development, and which does not produce a host cytokine. __

    Interesting comment but key novel result is that co-infection studies show reversed phenotype of IL-6 transgenic parasites, likely due to counteracting Of IL-6 effect by Wild type parasites (Supplementary Fig 1)

    __Another assumption is that IL-6 is secreted from the infected liver cell and mediates its effects, presumably by binding to its cell surface receptor. The expectation of Il-6 secretion from the parasite is that it would accumulate in the parasitophorous vacuole - how would it get out of the infected host cell? While evidence is provided of IL-6 in the in vitro culture supernatant of infected cells - this might arise from damaged cells in rather artificial conditions. Have the authors considered doing the experiment of concurrent mouse infection with both wild type and recombinant parasites? If the mechanism of parasite killing in infected liver cells is as proposed, then a reduction of wild type parasites in the subsequent asexual blood stage would be expected. __

    Experiments done. We discussed both experiments: IL-6 receptor blocking antibody experiement (Suppl Fig 2), and mixed infection (Suppl Fig 1).

    __Figure 3 indicates that IL-6 TgPbA/LISP2 parasites are as efficient or better than wild type parasites at invading host cells but then they do not develop to maturity. What is the evidence that the key factor in their ability to immunize the host is expression of IL-6 rather than the effect of an attenuated parasite? __

    This is an interesting observation made by the reviewer. With the available data, we cannot really tell which of the two possibilities is operating in thin system. It could also be that the two option are interconnected.

    __In this model malaria infection, it looks like there are two lethal outcomes: one associated with experimental cerebral malaria at relatively low blood stage parasitemia (which I understand is a controversial model for human cerebral malaria) and the second associated with high blood stage parasitemia. Some of the protocols affect which outcome occurs (see for example Fig 6), but this observation is not properly discussed. __

    In many occasions, we did see in the past a discrepancy between anti-parasite immunity and anti-disease protection. In this particular experiment (Fig 6), we explored the dose effect of the IL-6 mutant. What is clear from this model is that at the high dose, 104 SPZ, we observe both anti-parasite and anti-disease protection and immunity, whereas at the lower doses, 103 and 102 SPZ, although there was no efficient anti-parasite immunity, mice did not die from cerebral malaria but much later from hyperparasitemia. We consider that the two low doses of IL-6 transgenic parasites did protect against disease expression.

    __For the data presented in Fig 7, why was there a challenge with WT PbA sporozoites before the heterologous Py challenge? If this step is excluded is there still an effect against P. yoelii? Why was the parasite chosen for the heterologous challenge Py17XNL? Since this parasite is largely restricted to reticulocytes in the blood stream would a different effect have been observed if the heterologous challenge parasite was, for example, P. chabaudi? __

    Out of scope.

    __Although the expectation is that IL-6 expression would not occur in the asexual blood stage, I think it would be important to demonstrate experimentally that this is the case. __

    Done. IL-6 transgenic parasite, when inoculated as infected erythrocytes have no development defect and grow normally in infected mice.

    In Fig. 4A the y-axis is labelled IL-6 rRNA when it should be IL-6 mRNA.

    Corrected

    Reviewer #2 (Significance (Required)):

    __The significance of the report does depend on whether or not the experimental evidence is sufficient to support the claim that parasite expression of IL-6 is important in generating immunity. There has been a number of studies to show that infection with sporozoites that have been genetically attenuated to not complete subsequent development in the infected liver cell can provide immunity to subsequent infection; what is different about this study is that the authors specifically target the parasite to express a host protein that is likely to be important in acquisition of immunity. Therefore for the study to have high significance they have to show convincingly that it is the expression and activity of IL-6 that is important and I do not think this is the case with the experiments reported. If the authors are correct, then the idea of manipulating the host response by expression of host proteins by the parasite may be an attractive approach to dissect the key elements of immunity to sporozoite infection. At the moment, although there is a lot of focus on developing an attenuated whole sporozoite vaccine against malaria, and this study may provide proof of principle for including a host component in the parasite, there would still be long way to go before any practical application of this approach. __

    The key message was toned down. As the formal demonstration that the expression and activity of IL-6 is direcxtly involved in IL-6 transgenic parasites to confer protective immunity, we suggest to tone down the message by saying that IL-6 attenuates parasite virulence, the mechanism being likely through IL6 signaling detrimental effect on parasite development.

    The audience would be those interested in parasite immunology.

    __

    Reviewer expertise: malaria parasite cell and molecular biology; host immunity.

    **Referees cross commenting** __

    __ I think all reviewers are of the opinion that there needs to be a better demonstration that the observed phenotype is mediated by expression and signaling of IL-6, for example by antibody blockade or using a mouse line with a genetic defect in IL-6 signaling. Looking at all the issues that have been raised by the reviewers and need to be addressed with further experimentation, my feeling is that this will take longer than 6 months.

    __

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    __ **Summary** This study explores the expression of murine IL-6 by rodent Plasmodium berghei as a means to generate transgenic parasites whose development in the liver is arrested, which may be used as a genetically attenuated pre-erythrocytic vaccine against malaria. The authors conclude that IUL-6-expressing Plasmodium parasites elicit CSD8+ T-cell mediated immune responses that protect against a subsequent challenge with infectious sporozoites.

    **Major Comments** __

    __ In Figure 3, the authors show the results of qRT-PCR analysis of mouse livers infected with WT or transgenic parasites. They then use HepG2 cells to assess hepatic parasite numbers and development. Why didn't the authors assess this also in vivo, in liver sections of infected mice?__

    Done. New data are presented in Fig 3B, C, D

    Linked to the above, a more complete analysis of the parasite's behavior in HepG2 cells should be provided. The authors write in the discussion that "IL-6 transgenic parasites develop perfectly well in cultured hepatocytic cells". Does this mean that they develop to the production of infectious merozoites? This could be confirmed by allowing the infected cultures to progress for 60-70 hours and then collecting the supernatants of these cultures and injecting them into naïve mice, to understand whether or not infectious merozoites are formed in vitro.

    New analysis demonstrate that IL-6 transgenic parasites actually display a developmental defect at the pre-erythrocytic stage in vivo.

    __Figure 3C: The authors mention this result almost in passing but fail to provide an explanation for this observation. Why is the number of transgenic parasite EEFs approximately double that of WT parasite EEFs? __

    A new figure 3 is provided and show that the EEF density (Fig 3B) was drastically reduced both at 24h and at 48h in mice infected with the IL-6 transgenic parasites as compared to those infected with WT PbA parasites, although the differences were not statistically significant. We also examined the size (Fig. 3C) of EEFs, and found the same tendency, namely a reduced size and diameter of IL-6 transgenic EEF as compared to those of WT PbA EEFs with a statistical difference only at 40h.

    __Figure 3D: The EEF area units (mm2) on the YY axis are certainly wrong. However, they cannot be um2 either, as 15-30 um2 would be far too small for EEFs at 48 hours post-infection. What is it then? __

    New data are now provided in a new Fig 3.

    The authors write "... suggest that the failure of IL-6 Tg-PbANKA/LISP2 parasites to develop in the liver of infected mice is likely due to an active anti-parasite immune response mediated by parasite-encoded IL-6 in vivo". I have several issues with this statement. 1) as mentioned above, the in vitro data cannot be used to draw definitive conclusions about the parasites' behavior in vivo; 2) the transgenic parasites do not "fail to develop in the liver of infected mice". If anything, they develop less than their WT counterparts, which is different from "failing to develop". Clarifying how much they do develop would be important (see next comment).

    We provide new in vivo data as to the development of IL-6 transgenic parasites. A new figure 3 is provided and show that the EEF density (Fig 3B) was drastically reduced both at 24h and at 48h in mice infected with the IL-6 transgenic parasites as compared to those infected with WT PbA parasites, although the differences were not statistically significant. We also examined the size (Fig. 3C) of EEFs, and found the same tendency, namely a reduced size and diameter of IL-6 transgenic EEF as compared to those of WT PbA EEFs with a statistical difference only at 40h. We replaced failure by a defect in development.

    In connection with the above, I would like to know more about the time when the development of IL-6 Tg-PbANKA/LISP2 parasites is arrested in vivo, in the liver. Are these early- or late-arresting parasites? Is the liver stage of infection compromised during parasite development or at egress? To clarify this, the manuscript would benefit from a timecourse analysis of liver sections of mice infected with this parasite, including data on EEF numbers and sizes up to and beyond 48 h after sporozoite inoculation.

    Done. See new figure 3.

    __Still linked to the issue of parasite arrest in vivo and the possibility of breakthroughs, the manuscript would benefit from an experiment where mice were injected with a high number of transgenic sporozoites and parasitemia is monitored thereafter, much like what was done in Figure 2D, but starting off with a larger inoculum of at least 5 x 10^5 sporozoites. __

    This was done and there was no breakthrough even with doses as high as 106 sporozoites

    While the results shown to suggest that secreted IL-6 restricts the parasite's liver stage development in vivo, this could be more definitely demonstrated by performing an infection with the transgenic parasites in the context of blocking or absence of the host's IL-6 receptor. This experiment was done but unfortunately did not work (Suppl. Fig 2). That is, the treatment of mice infected with IL-6 transgenic parasites with anti-IL-6 receptor blocking antibodies did not reverse the infection phenotype. This was also discuss in the manuscript.

    **Minor Comments**

    __

    The manuscript needs to be improved in terms of both language and format. Some examples, solely from the abstract, are listed below, but the manuscript needs to be appropriately revised in terms of language, grammar, punctuation and format throughout:__

    __-Space missing between "P." and "berghei" __

    Done

    __-Gene names should be italicized __

    Done

    -Rephrase "Considering IL-6 as a critical proinflammatory signal..." to "Considering that IL-6 is a critical proinflammatory signal..."

    Done

    -"transgenic IL-6 sporozoites" should be "transgenic IL-6-expressing P. berghei sporozoites"

    Done

    -"impairs Plasmodium infection at the liver stage" should be "impairs the liver stage of Plasmodium infection"

    Done

    INTRODUCTION

    The sentence "Among them, parasites lacking integrity of the parasitophorous vacuole, or late during development, and..." appears to be incomplete and needs rephrasing.

    Done

    The references used in sentence "During the last decade, in search of key mechanisms that determine the host inflammatory response, a set of host factors turned out to be critical for malaria parasite liver stage development (Mathieu et al., 2015); (Demarta-Gatsi et al., 2017; Demarta-Gatsi et al., 2016) (Grand et al., 2020)" do not all relate to the liver stage of infection. The authors need to select references that are relevant for their statement or else change the statement.

    Rephrased

    RESULTS

    I suggest the authors change the title of Results section "Transgenic P. berghei parasites expressing IL-6 during the liver stage lose infectivity to mice" not only to improve the quality of the English language employed but also to better clarify the notion that they are talking about hepatic infectivity.

    __On the same section, please correct "timely specific timely". __

    Done

    Transfectants are not "verified". If anything, the insertion of the gene in the parasite's genome is verified or, better still, confirmed.

    Done

    Sentence "The two lines behave similarly" is redundant.

    Done

    The legend of Figure 1 must include the definitions of all the acronyms in that figure.

    Acronyms in the whole manuscript are defined elsewhere

    __"IL-6 transgenic sporozoites" is not an appropriate designation. If anything, they should be called IL-6-expressing P. berghei sporozoites". __

    Done

    Figure 2 B: The YY axis should clarify that it refers to sporozoite numbers, as there are many other parasite stages in mosquitoes.

    Done

    Figure 2C: This scheme is hardly necessary. It would suffice to label the plots in D and E with the names of the parasite lines employed rather than "Group 1", "Group 2", "Group 3". The scheme is provided for more clarity and easy reading of the accompanying figures

    Figure 2D, 2E: Why didn't the authors use the same scale on the XX axis of the two plots?

    The qRT-PCR data per se do not substantiate the statement "Therefore, RT-qPCR analysis in the liver confirms that the loss of infectivity of IL-6 Tg-PbANKA/LISP2 SPZ is due to a defect in liver stage development in vivo", as a defect in invasion of hepatocytes cannot be excluded. The term "loss of infectivity" is also misleading. Do the authors mean loss of blood stage infectivity?

    Yes

    Sentence "... all parasites were able to invade and develop inside HepG2 cells." is misleading. The authors probably mean "parasites of both lines".

    Changed

    Figure 4: Why did the authors swap the order of the two experimental groups from one plot to the next? The same order should be used, to avoid confusion! Also, the authors should make the width of the bars in similar between the two plots.

    Done

    The authors should consider moving Figure 5 to the Supplementary materials.

    Reviewer #3 (Significance (Required)):

    *Nature and significance of the advance. Compare to existing published knowledge. Audience.*

    This study extends our current knowledge on genetically attenuated malaria vaccine candidates and validates the concept of suicide parasites for immunization against malaria. This paper will be of interest to researchers working on malaria vaccination, as well as all those interested in transgenic Plasmodium parasites, and the biology and immunology of liver stage infection by malaria parasites.

    *Your expertise.*

    The co-reviewer and the reviewer are experts on the liver stage of Plasmodium infection and on pre-erythrocytic malaria vaccination.

    **Referees cross commenting**

    I agree with all of Reviewers 1 and 2's remarks and, upon consideration, I would like to revise my "Estimated time to Complete Revisions" to become between 3 and 6 months

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    This study explores the expression of murine IL-6 by rodent Plasmodium berghei as a means to generate transgenic parasites whose development in the liver is arrested, which may be used as a genetically attenuated pre-erythrocytic vaccine against malaria. The authors conclude that IUL-6-expressing Plasmodium parasites elicit CSD8+ T-cell mediated immune responses that protect against a subsequent challenge with infectious sporozoites.

    Major Comments

    In Figure 3, the authors show the results of qRT-PCR analysis of mouse livers infected with WT or transgenic parasites. They then use HepG2 cells to assess hepatic parasite numbers and development. Why didn't the authors assess this also in vivo, in liver sections of infected mice?

    Linked to the above, a more complete analysis of the parasite's behavior in HepG2 cells should be provided. The authors write in the discussion that "IL-6 transgenic parasites develop perfectly well in cultured hepatocytic cells". Does this mean that they develop to the production of infectious merozoites? This could be confirmed by allowing the infected cultures to progress for 60-70 hours and then collecting the supernatants of these cultures and injecting them into naïve mice, to understand whether or not infectious merozoites are formed in vitro.

    Figure 3C: The authors mention this result almost in passing but fail to provide an explanation for this observation. Why is the number of transgenic parasite EEFs approximately double that of WT parasite EEFs?

    Figure 3D: The EEF area units (mm2) on the YY axis are certainly wrong. However, they cannot be um2 either, as 15-30 um2 would be far too small for EEFs at 48 hours post-infection. What is it then?

    The authors write "... suggest that the failure of IL-6 Tg-PbANKA/LISP2 parasites to develop in the liver of infected mice is likely due to an active anti-parasite immune response mediated by parasite-encoded IL-6 in vivo". I have several issues with this statement. 1) as mentioned above, the in vitro data cannot be used to draw definitive conclusions about the parasites' behavior in vivo; 2) the transgenic parasites do not "fail to develop in the liver of infected mice". If anything, they develop less than their WT counterparts, which is different from "failing to develop". Clarifying how much they do develop would be important (see next comment).

    In connection with the above, I would like to know more about the time when the development of IL-6 Tg-PbANKA/LISP2 parasites is arrested in vivo, in the liver. Are these early- or late-arresting parasites? Is the liver stage of infection compromised during parasite development or at egress? To clarify this, the manuscript would benefit from a timecourse analysis of liver sections of mice infected with this parasite, including data on EEF numbers and sizes up to and beyond 48 h after sporozoite inoculation.

    Still linked to the issue of parasite arrest in vivo and the possibility of breakthroughs, the manuscript would benefit from an experiment where mice were injected with a high number of transgenic sporozoites and parasitemia is monitored thereafter, much like what was done in Figure 2D, but starting off with a larger inoculum of at least 5 x 10^5 sporozoites.

    While the results shown to suggest that secreted IL-6 restricts the parasite's liver stage development in vivo, this could be more definitely demonstrated by performing an infection with the transgenic parasites in the context of blocking or absence of the host's IL-6 receptor.

    Minor Comments

    The manuscript needs to be improved in terms of both language and format. Some examples, solely from the abstract, are listed below, but the manuscript needs to be appropriately revised in terms of language, grammar, punctuation and format throughout:

    -Space missing between "P." and "berghei"

    -Gene names should be italicized

    -Rephrase "Considering IL-6 as a critical proinflammatory signal..." to "Considering that IL-6 is a critical proinflammatory signal..."

    -"transgenic IL-6 sporozoites" should be "transgenic IL-6-expressing P. berghei sporozoites"

    -"impairs Plasmodium infection at the liver stage" should be "impairs the liver stage of Plasmodium infection"

    INTRODUCTION

    The sentence "Among them, parasites lacking integrity of the parasitophorous vacuole, or late during development, and..." appears to be incomplete and needs rephrasing.

    The references used in sentence "During the last decade, in search of key mechanisms that determine the host inflammatory response, a set of host factors turned out to be critical for malaria parasite liver stage development (Mathieu et al., 2015); (Demarta-Gatsi et al., 2017; Demarta-Gatsi et al., 2016) (Grand et al., 2020)" do not all relate to the liver stage of infection. The authors need to select references that are relevant for their statement or else change the statement.

    RESULTS

    I suggest the authors change the title of Results section "Transgenic P. berghei parasites expressing IL-6 during the liver stage lose infectivity to mice" not only to improve the quality of the English language employed but also to better clarify the notion that they are talking about hepatic infectivity.

    On the same section, please correct "timely specific timely".

    Transfectants are not "verified". If anything, the insertion of the gene in the parasite's genome is verified or, better still, confirmed.

    Sentence "The two lines behave similarly" is redundant.

    The legend of Figure 1 must include the definitions of all the acronyms in that figure.

    "IL-6 transgenic sporozoites" is not an appropriate designation. If anything, they should be called IL-6-expressing P. berghei sporozoites".

    Figure 2 B: The YY axis should clarify that it refers to sporozoite numbers, as there are many other parasite stages in mosquitoes.

    Figure 2C: This scheme is hardly necessary. It would suffice to label the plots in D and E with the names of the parasite lines employed rather than "Group 1", "Group 2", "Group 3".

    Figure 2D, 2E: Why didn't the authors use the same scale on the XX axis of the two plots?

    The qRT-PCR data per se do not substantiate the statement "Therefore, RT-qPCR analysis in the liver confirms that the loss of infectivity of IL-6 Tg-PbANKA/LISP2 SPZ is due to a defect in liver stage development in vivo", as a defect in invasion of hepatocytes cannot be excluded. The term "loss of infectivity" is also misleading. Do the authors mean loss of blood stage infectivity?

    Sentence "... all parasites were able to invade and develop inside HepG2 cells." is misleading. The authors probably mean "parasites of both lines".

    Figure 4: Why did the authors swap the order of the two experimental groups from one plot to the next? The same order should be used, to avoid confusion! Also, the authors should make the width of the bars in similar between the two plots.

    The authors should consider moving Figure 5 to the Supplementary materials.

    Significance

    Nature and significance of the advance. Compare to existing published knowledge. Audience.

    This study extends our current knowledge on genetically attenuated malaria vaccine candidates and validates the concept of suicide parasites for immunization against malaria. This paper will be of interest to researchers working on malaria vaccination, as well as all those interested in transgenic Plasmodium parasites, and the biology and immunology of liver stage infection by malaria parasites.

    Your expertise.

    The co-reviewer and the reviewer are experts on the liver stage of Plasmodium infection and on pre-erythrocytic malaria vaccination.

    Referees cross commenting

    I agree with all of Reviewers 1 and 2's remarks and, upon consideration, I would like to revise my "Estimated time to Complete Revisions" to become between 3 and 6 months

  5. 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

    The manuscript describes the construction of a Plasmodium berghei that expresses murine interleukin-6 in exoerythrocytic (liver stage) parasites and the analysis of mice infected with sporozoites of this parasite line. They find that such parasites do not complete development in liver cells and therefore do not produce subsequent infection in red blood cells. The ability of prior infection with these parasites on the ability of the host to resist both wild type and heterologous species challenge is then examined.

    The key assumption that underlies the study is that the observed phenotypes result from parasite expression of bioactive IL-6 that functions to modulate the immune system. Other explanations are not considered, for example the over-expression of secreted IL-6 may prevent the complete maturation of the intracellular parasite by clogging up the parasite secretory pathway. The authors use the 'wild type' parasite as the control but not only does the wild type not express IL-6 it also does not express the human DHFR gene used as a selection system. A much better control parasite would be one that expresses a non-bioactive IL-6 so that the potential effects on parasite maturation can be differentiated from those on the mouse immune system. Another control to be considered would be comparison with a genetically attenuated parasite with a block in late stage development, and which does not produce a host cytokine.

    Another assumption is that IL-6 is secreted from the infected liver cell and mediates its effects, presumably by binding to its cell surface receptor. The expectation of Il-6 secretion from the parasite is that it would accumulate in the parasitophorous vacuole - how would it get out of the infected host cell? While evidence is provided of IL-6 in the in vitro culture supernatant of infected cells - this might arise from damaged cells in rather artificial conditions. Have the authors considered doing the experiment of concurrent mouse infection with both wild type and recombinant parasites? If the mechanism of parasite killing in infected liver cells is as proposed, then a reduction of wild type parasites in the subsequent asexual blood stage would be expected.

    Figure 3 indicates that IL-6 TgPbA/LISP2 parasites are as efficient or better than wild type parasites at invading host cells but then they do not develop to maturity. What is the evidence that the key factor in their ability to immunize the host is expression of IL-6 rather than the effect of an attenuated parasite?

    In this model malaria infection, it looks like there are two lethal outcomes: one associated with experimental cerebral malaria at relatively low blood stage parasitemia (which I understand is a controversial model for human cerebral malaria) and the second associated with high blood stage parasitemia. Some of the protocols affect which outcome occurs (see for example Fig 6), but this observation is not properly discussed.

    For the data presented in Fig 7, why was there a challenge with WT PbA sporozoites before the heterologous Py challenge? If this step is excluded is there still an effect against P. yoelii? Why was the parasite chosen for the heterologous challenge Py17XNL? Since this parasite is largely restricted to reticulocytes in the blood stream would a different effect have been observed if the heterologous challenge parasite was, for example, P. chabaudi?

    Although the expectation is that IL-6 expression would not occur in the asexual blood stage, I think it would be important to demonstrate experimentally that this is the case.

    In Fig. 4A the y-axis is labelled IL-6 rRNA when it should be IL-6 mRNA.

    Significance

    The significance of the report does depend on whether or not the experimental evidence is sufficient to support the claim that parasite expression of IL-6 is important in generating immunity. There has been a number of studies to show that infection with sporozoites that have been genetically attenuated to not complete subsequent development in the infected liver cell can provide immunity to subsequent infection; what is different about this study is that the authors specifically target the parasite to express a host protein that is likely to be important in acquisition of immunity. Therefore for the study to have high significance they have to show convincingly that it is the expression and activity of IL-6 that is important and I do not think this is the case with the experiments reported. If the authors are correct, then the idea of manipulating the host response by expression of host proteins by the parasite may be an attractive approach to dissect the key elements of immunity to sporozoite infection. At the moment, although there is a lot of focus on developing an attenuated whole sporozoite vaccine against malaria, and this study may provide proof of principle for including a host component in the parasite, there would still be long way to go before any practical application of this approach.

    The audience would be those interested in parasite immunology.

    Reviewer expertise: malaria parasite cell and molecular biology; host immunity.

    Referees cross commenting

    I think all reviewers are of the opinion that there needs to be a better demonstration that the observed phenotype is mediated by expression and signaling of IL-6, for example by antibody blockade or using a mouse line with a genetic defect in IL-6 signaling. Looking at all the issues that have been raised by the reviewers and need to be addressed with further experimentation, my feeling is that this will take longer than 6 months.

  6. 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 #1

    Evidence, reproducibility and clarity

    Whole sporozoite vaccines confer sterilizing protection against Plasmodium infection. However, further improvements of whole sporozoite vaccines is needed and requires a thorough understanding of the immune processes that mediate protection and the deployment of novel strategies further augment protective immunity while limiting the impact of factors that are detrimental to protection. Work from the Mecheri laboratory and others had previously established that IL-6 signaling plays a critical role in the immune response to a liver stage infection; engagement of IL-6 signaling promotes the initial control of a liver stage infection and enhances the protective adaptive immune response. Given this potent protective role for IL-6, Belhimeur and colleagues design a parasite strain in rodent malaria parasites that encodes and secrete murine IL-6 during liver stage infection. They show that upon infection of wildtype mice, these transgenic parasites i) are unable to transition to blood stage infection, ii) produce Il-6 and iii) induce a durable adaptive immune response that can protect against sporozoite challenge. This study is novel and intriguing. However, a superficial analysis of the transgenic parasite strain, an incomplete analysis of the immune response to infection and the lack of data regarding the possibility of IL-6 mediated immunopathology have dampened this reviewer's enthusiasm for the work.

    Major Concerns:

    1)The data in Figure 3b-3d clearly indicate that the IL-6 encoding transgenic parasites exhibit a defect in parasite development within HepG2 cells that is maintained in vivo. The authors propose that an arrest of these parasites in the liver stage precludes their transition to blood stage infection and that this arrest is dependent on IL-6 signaling. To better support that claim the authors should:

    a.Better characterize in vivo liver stage arrest using infected liver tissue analysis with immunofluorescence microscopy to determine when and how precisely IL-6 transgenic parasites are impacted in development.

    b.Determine if arrested development of IL-6 transgenic parasites is truly dependent on IL-6 signaling using antibody blockade of IL-6 signaling and mice with genetic defects in IL-6 signaling.

    2)The authors claim that IL-6 production and secretion into the liver tissue augments the adaptive immune response to liver stage infection. This in turn results in a durable adaptive immune responses that protect against infection. However, the mechanistic underpinning of IL-6 signaling in the liver that is induced by their transgenic parasites and the impact on adaptive immune responses is poorly characterized:

    a.There is no evidence that the protective adaptive immune response induced by IL-6 trangenic parasite infection is dependent on IL-6 signaling. Is superior protection and immunogenicity lost in IL-6 signaling deficient animals that are infected with IL-6 transgenic parasites?

    b.What elements of the adaptive immune response are impacted? One can imagine that IL-6 mediated killing of infected hepatocytes might introduce more parasite antigen that can be acquired by antigen presenting cells, or that IL-6 mediated pro-inflammatory signaling might regulate the maturation of antigen presenting cells, increased differentiation of helper T cells, the downregulation of regulatory T cell function and frequency and/or the differentiation of effector CD8 T cells into long-lived hepatic memory CD8 T cells. The authors should conduct a more comprehensive analysis of how parasite-encoded IL-6 impacts adaptive immunity.

    3)While IL-6 transgenic parasites induce a potent and durable adaptive immune response, the authors should show how this compares to published whole sporozoite immunizations. The authors should determine if immunization with IL-6 transgenic parasites is superior to for example immunization with radiation-attenuated sporozoites and generically attenuated sporozoites.

    1. IL-6 signaling is a major player in inflammatory diseases and the induction of immunopathology. As such the authors should carefully examine the duration and magnitude of IL-6 protein production in the liver, and serum after IL-6 Tg parasite infection and determine if IL-6 signaling promotes liver immunopathology.

    Significance

    The paper is reporting a novel strategy to generate a whole sporozoite vaccine. Expression of IL6 in a transgenic parasite. This could be a significant contribution to the field if additional experiments as outlined in the critique are conducted.The work might also inform vaccine design for other pathogens.

  7. Version published to 10.1101/2021.11.16.468835v1 on bioRxiv