From the journal RSC Chemical Biology Peer review history

10-Feb-2022

Dear Prof Compton:

Manuscript ID: CB-ART-12-2021-000251
TITLE: 6’’-Modifed α-GalCer-peptide conjugate vaccines protect against liver-stage malaria

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary. In particular, the reviewers raised some concerns about the in vivo work which requires additional revisions/experiments.

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Associate Editor, RSC Chemical Biology

************

Reviewer 1

The work submitted to RSC ChemBio entitled 6’’-Modifed α-GalCer-peptide conjugate vaccines protect against liver-stage malaria, by the group of B.J. Compton, describes the synthesis and in vivo studies (mice) of innovative α-GalCer conjugate vaccine candidate with a potential application against liver-stage malaria.
The work is a multidisciplinary collaboration by different groups and the chemical synthesis is supported by in vivo results. The pro-drug kind of approach, especially by using p-aminobenzyl linker to release in vivo the thiol-containing “drug” is very interesting. The work is well written, with a nice flow, complete literature and supported by different extra information in the supporting material.
At this stage the work in my opinion requires a major revision mostly due to the small animal work (n=3 with pooled data) and the way of presenting data. I do not ask the authors to perform more animal experiments, by I do ask to authors to answer to some of my main concerns to have a complete work for RSC ChemBiol:
1. Please among all the manuscript from the title to all the work, the authors should change the word vaccine with vaccine-candidate.
2. In the Abstract specify the Plasmodium studied and please do not use acronyms (GLP…)
3. Page 2, second paragraph, while describing the decision to functionalize 6’’ add the reference with the X-ray structure mentioned (18 later in the work).
4. Page 2, second paragraph, O to N migration with co-release of the active constituent, please add a reference.
5. Page 2, after listing references 19-29, please add the diseases approached in the mentioned studies.
6. Page 2, end of the intro, the authors did not prove the ability to protect against Plasmodium challenge, but a decrease in parasitemia. Please, the authors should me softer.
7. Cathepsin B has been mostly described as Cysteine protease and the authors did perform the enzymatic studies with the non-final product, i.e. the unconjugated molecule. Can the authors add the explanation for that in the text linking this to the peptide structure used as model?
8. Scheme-4, the authors should mention in the next the presence of ST between FFRKAAA and NVFDNNL.
9. Why did the authors select the intravenous route compared to the other ones (im or ip)?
10. Important point: due to the solubility issues and last purification steps, the authors should perform a BCA or uBCA assay to the products they are comparing to be sure about the 0.135 nmoles/200uL PBS. Doing this only gravimetrically, especially while comparing similar structures could affect drastically the in vivo results.
11. Why pooled samples and not individual ones? Do the authors also have the individual results? Do the authors only see these effects while pooling the samples? The authors should describe and mention all the data they have.
12. Figure 3, the authors should show the statistical analysis column per column.
13. Figure 3, and also the other in vivo experiments, why the authors do not have a control with the peptide without the α-GalCer? Do they have preliminary results proving that the peptide per se in not immunogenic in these conditions?
14. Figure 3 SI, E-F, The authors should add the result with the naïve mice.
15. Is Nv the abbreviation for naïve? This is not clear in the text
16. Why did the authors not use a placebo group injected with PBS?
17. Figure 5B, the data with 200spz and 3000spz should be represented independently at least in the supporting information

Reviewer 2

The manuscript by Meijlink et al describes a synthetic route for the creation and pre-clinical in-vivo validation of a new self-adjuvating GLP conjugate vaccine against pre-erythrocytic malaria. The authors use an elegant synthesis process to create two pro-adjuvants, the 6’’-deoxy-6’’-thio and amino analogues of α-GalCer, which were fused by a cathepisin B-cleavable linker to a short peptide epitope identified from the Plasmodium berghei (Pb) ribosomal protein, PbRPL6. These vaccine candidates were analyzed for their capacity to induce NKT and CD8 T cell activation in mice livers after a single intravenous immunization, and to generate sterile protection against a new infection. The authors conclude that the 6’’-azido-6’’deoxy- α-GalCer conjugate candidate induces a significantly higher number of specific liver CD8+ Trm cells and a stronger activation of NKT cells in the liver and the spleen of immunized animals comparatively to a previously published GLP-conjugate vaccine candidate generated by the same group. Furthermore, this candidate warrants the sterile protection of all immunized mice against a challenge and a rechallenge with Pb sporozoites, 30 and 51 days after immunization, respectively.
Overall, the rationale for the study is clearly explained, well documented and logically given in perspective. The authors use a carefully planned methodology that is adapted to the finality of the demonstrations and clearly described. The figures correctly display the results, allowing their clear interpretation and discussion. The manuscript is well written and provides adequate literature citations.
As a minor point, I question the choice of a low dosage of 200 spz to perform the 30 day challenge of immunized animals. Taking into account the well-established practice of at least 5 mosquito bites for obtaining a successful CHMI challenge of 100% of the volunteers and that the established range of sporozoites injected by each mosquito is 50-500, the use of 200 sporozoites is clearly on the lower end of the threshold to warranty infection of all mice. Even though, the route of challenge is different and all non-immunized control mice became infected, it still seems to this reviewer that such low challenge dosage may be potentially overestimating the protective efficacy of the vaccine candidate. A later rechallenge of the previously protected mice with a higher dosage does not necessary addresses this, as you may consider that the killing parasites of the first challenge may work to boost the previously induced vaccine immune response. I would therefore suggest the authors to use a higher dosage of sporozoites to challenge immunized mice, of at least 1000 spz or even higher.
In summary, I believe that the study regards an important biological topic, leading to broadly compelling and relevant results that are suited for the readership of RSC Chemical Biology.

We kindly thank the Editor and Reviewers for the effort they have put into reviewing this manuscript. We have addressed all of the reviewers concerns which are documented in the attached files (in red text) next the original reviewer comment.

This text has been copied from the Word file response to reviewers and does not include any figures, images or special characters:

REVIEWER REPORT(S):
Referee: 1

Comments to the Author
The work submitted to RSC ChemBio entitled 6’’-Modifed α-GalCer-peptide conjugate vaccines protect against liver-stage malaria, by the group of B.J. Compton, describes the synthesis and in vivo studies (mice) of innovative α-GalCer conjugate vaccine candidate with a potential application against liver-stage malaria.
The work is a multidisciplinary collaboration by different groups and the chemical synthesis is supported by in vivo results. The pro-drug kind of approach, especially by using p-aminobenzyl linker to release in vivo the thiol-containing “drug” is very interesting. The work is well written, with a nice flow, complete literature and supported by different extra information in the supporting material.
At this stage the work in my opinion requires a major revision mostly due to the small animal work (n=3 with pooled data) and the way of presenting data. I do not ask the authors to perform more animal experiments, by I do ask to authors to answer to some of my main concerns to have a complete work for RSC ChemBiol:

1. Please among all the manuscript from the title to all the work, the authors should change the word vaccine with vaccine-candidate.

The authors respectfully disagree with the reviewer that the glycolipid-peptide conjugates discussed in this work should be called “vaccines-candidates” and not the more widely-used term for these constructs (see selected References 1-5) which is just simply “vaccines.” Our glycolipid-peptide conjugates fit with Mariam-Webster’s (an all other common dictionaries) definition of the word “vaccine” which is generally accepted as “a preparation that is administered (as by injection) to stimulate the body's immune response against a specific infectious agent or disease.” It is not clear to the authors as what is gained or is more enlightening to the readers by calling these substances “vaccine-candidates” and not simply “vaccines.”

2. In the Abstract specify the Plasmodium studied and please do not use acronyms (GLP…)

“Plasmodium berghei ANKA (PbA)” was included in the abstract and “GLP-vaccines” was replaced with “conjugate vaccines”

3. Page 2, second paragraph, while describing the decision to functionalize 6’’ add the reference with the X-ray structure mentioned (18 later in the work).

The reference: Borg, N. A. et al. Nature 2007, 448 (7149), 44-49 has been added.

4. Page 2, second paragraph, O to N migration with co-release of the active constituent, please add a reference.

The reference: Anderson, R. J. et al. Nat Chem Biol 2014, 10 (11), 943-949 has been added.

5. Page 2, after listing references 19-29, please add the diseases approached in the mentioned studies.

Most of the referenced papers do not explore the role of 6’’-modified α-GalCer analogues in disease models but instead often measure the cytokines produced allowing the compounds to be defined as either skewed towards Th1, Th2, or remain unbiased (compared to α-GalCer). To more thoroughly address the biological responses of the mentioned compounds, the authors have added “…..many of which display potent immunostimulatory activities that are biased for either a Th16-13 or Th26-7, 14 response compared to α-GalCer.” to the main text.

6. Page 2, end of the intro, the authors did not prove the ability to protect against Plasmodium challenge, but a decrease in parasitemia. Please, the authors should me softer.

We respectfully disagree with the referee on this point as we clearly show that our vaccines protect against a sporozoite challenge. In this work we measured protection by examining parasitemia and show that that parasitemia in protected mice was not decreased, it was absent, indicating protection.

7. Cathepsin B has been mostly described as Cysteine protease and the authors did perform the enzymatic studies with the non-final product, i.e. the unconjugated molecule. Can the authors add the explanation for that in the text linking this to the peptide structure used as model?

Have changed the paragraph to read “Prior to the synthesising the full GLP-vaccines, the susceptibility of the PAB-Cit-Val linker to human liver-derived cathepsin B was investigated in a simplified system whereby the pro-adjuvant 18 was exposed to the enzyme for 24 h at 37 °C in a mixture of HFIP/DMSO in ammonium acetate buffer.”

8. Scheme-4, the authors should mention in the next the presence of ST between FFRKAAA and NVFDNNL.

Have changed the paragraph to read: “Here, the CD8 epitope was adorned with naturally flanking amino acid residues -ST- (N-terminus) and -S (C-terminus) from the protein antigen and the N-terminal -FFRK- proteasomal cleavage sequence, separated by a tri-alanine (-AAA-) spacer.”

9. Why did the authors select the intravenous route compared to the other ones (im or ip)?

The i.v. route is used maximise access to secondary lymphoid tissue (i.e. the spleen and liver) where NKT cell numbers are high.

10. Important point: due to the solubility issues and last purification steps, the authors should perform a BCA or uBCA assay to the products they are comparing to be sure about the 0.135 nmoles/200uL PBS. Doing this only gravimetrically, especially while comparing similar structures could affect drastically the in vivo results.

Although the pro-adjuvant compounds can suffer from solubility issues, the actual GLP-vaccines are readily soluble in organic solvents including DMSO (~20 mg/mL). The purity of the vaccine candidates was evaluated using LCMS-CAD, a technique well established for the both the quantification and qualification of organic compounds such as those produced in this work. The authors now realise that they failed to include how the vaccines were solubilised prior to administration for this body of work. To address this shortcoming the following statement has been added to the Supplementary Methods in the Supporting Information:

“Vaccination:
Solubilization of vaccines 26-28 was achieved by freeze-drying the samples in the presence of sucrose, L-histidine and Tween 20 as previously described for the solubilization of -GalCer.15 The solubilized compounds were diluted with phosphate-buffered saline (PBS) and mice were intravenously injected with 0.135 nmol of conjugate vaccines in 200µL PBS.”

Unfortunately, due to the concentrations of all the components in the solubilized vaccines, the BCA, Micro BCA, and CBQCA assays are not suitable. When ready for injection, the reconstituted vaccine contains:
• API – 0.005 mg/mL
• Tween 20 – 0.61 mg/mL
• Histidine – 0.92 mg/mL
• Sucrose – 0.67 mg/mL
BCA: According to the compatibility chart found on the ThermoFisher website (https://www.thermofisher.com/order/catalog/product/23227?SID=srch-srp-23227), the BCA assay requires:
• API range at 20-2000 µg/mL
• For a 1000 µg/mL sample,
o Tween 20 ≤5%
o Sucrose ≤40%
The concentration of our conjugate at injection concentration is only 5 µg/mL, so it is too dilute for this assay. Tween 20 and sucrose amounts in our formulation exceeds the allowed amounts for the assay.
Micro BCA: According to the compatibility chart found on the ThermoFisher website (https://www.thermofisher.com/order/catalog/product/23235), the micro BCA assay requires:
• API range at 0.5-20 µg/mL
• For a 10 µg/mL sample,
o Tween 20 ≤5%
o Sucrose ≤4%
Tween 20 and sucrose amounts in our formulation exceeds the allowed amount for the assay.
CBQCA: According to the document describing the assay on the ThermoFisher website (https://www.thermofisher.com/order/catalog/product/C6667), the CBQCA assay requires:
• API range of more than 10ng/mL protein
• In the sample,
o Tween 20 ≤0.1%
o Sucrose ≤10%
o Amines should be avoided
The histidine in the sample would severely interfere with the analysis. The amounts of Tween 20 and sucrose exceeds the amount allowed for the assay and our vaccines contain amino groups.

11. Why pooled samples and not individual ones? Do the authors also have the individual results? Do the authors only see these effects while pooling the samples? The authors should describe and mention all the data they have.

To clarify, the biological results presented in the manuscript are from two independent experiments that are combined into the same Figure. It is routine in our lab to plan experiments with 3-4 mice per group which are repeated before statistical analysis. This usually provides enough power to detect relevant differences. We look for trends in individual experiments to ensure that they appear to give similar results before pooling and assessing statistical significance between groups. Without pooling, numbers of mice in individual experiments may be too small to necessarily give significant results, so significance is not assessed before pooling. As liver Trm cell numbers are primarily responsible for protection,16 we show individual data for Trm for each mouse on our graphs so others can see the individual data variation. For completeness, we also show all memory T cell populations in the spleen and liver in a simpler column graph with mean and SE for each T cell type – to provide an overall picture of T cell immunity. To address the reviewer’s issue, we have provided all individual mouse data points for Figures 3, 4 and 5 (below).




12. Figure 3, the authors should show the statistical analysis column per column.

Have amended Fig 3 (below) to show this and have amended the legend accordingly.

13. Figure 3, and also the other in vivo experiments, why the authors do not have a control with the peptide without the α-GalCer? Do they have preliminary results proving that the peptide per se in not immunogenic in these conditions?

Peptide is well known to induce tolerance if injected alone.17 Although we have not used peptide alone here, we have previously shown that simply combining peptide with α-GalCer did not generate liver Trm.18

14. Figure 3 SI, E-F, The authors should add the result with the naïve mice.

Have amended SI Fig 3 E/F (below) to show this and have amended the legend accordingly.

We now also show the naïve control in Fig 4 A-H (below) in addition to the statistics for each column. The Figure legends have been amended accordingly.

15. Is Nv the abbreviation for naïve? This is not clear in the text.

Yes. This is abbreviation is now included in the appropriate figure legends.

16. Why did the authors not use a placebo group injected with PBS?

As in our response to 13 (above) we have previously shown that peptide alone or admixed with α-GalCer does not induce Trm so we chose not to inject PBS as the control and instead use naïve mice. As seen for naïve mice - now shown in Figures 4G/H and 5A - there is essentially no response without priming with an efficacious vaccine. We would not expect PBS to change these numbers, so deemed it unnecessary to subject mice to additional unnecessary injections.

17. Figure 5B, the data with 200spz and 3000spz should be represented independently at least in the supporting information.

Have included SI Fig 4A and B (below) to show the 200spz and 3000spz results independently.


Referee: 2

Comments to the Author
The manuscript by Meijlink et al describes a synthetic route for the creation and pre-clinical in-vivo validation of a new self-adjuvating GLP conjugate vaccine against pre-erythrocytic malaria. The authors use an elegant synthesis process to create two pro-adjuvants, the 6’’-deoxy-6’’-thio and amino analogues of α-GalCer, which were fused by a cathepisin B-cleavable linker to a short peptide epitope identified from the Plasmodium berghei (Pb) ribosomal protein, PbRPL6. These vaccine candidates were analyzed for their capacity to induce NKT and CD8 T cell activation in mice livers after a single intravenous immunization, and to generate sterile protection against a new infection. The authors conclude that the 6’’-azido-6’’deoxy- α-GalCer conjugate candidate induces a significantly higher number of specific liver CD8+ Trm cells and a stronger activation of NKT cells in the liver and the spleen of immunized animals comparatively to a previously published GLP-conjugate vaccine candidate generated by the same group. Furthermore, this candidate warrants the sterile protection of all immunized mice against a challenge and a rechallenge with Pb sporozoites, 30 and 51 days after immunization, respectively. Overall, the rationale for the study is clearly explained, well documented and logically given in perspective. The authors use a carefully planned methodology that is adapted to the finality of the demonstrations and clearly described. The figures correctly display the results, allowing their clear interpretation and discussion. The manuscript is well written and provides adequate literature citations. In summary, I believe that the study regards an important biological topic, leading to broadly compelling and relevant results that are suited for the readership of RSC Chemical Biology.

1. As a minor point, I question the choice of a low dosage of 200 spz to perform the 30 day challenge of immunized animals. Taking into account the well-established practice of at least 5 mosquito bites for obtaining a successful CHMI challenge of 100% of the volunteers and that the established range of sporozoites injected by each mosquito is 50-500, the use of 200 sporozoites is clearly on the lower end of the threshold to warranty infection of all mice. Even though, the route of challenge is different and all non-immunized control mice became infected, it still seems to this reviewer that such low challenge dosage may be potentially overestimating the protective efficacy of the vaccine candidate. A later rechallenge of the previously protected mice with a higher dosage does not necessary addresses this, as you may consider that the killing parasites of the first challenge may work to boost the previously induced vaccine immune response. I would therefore suggest the authors to use a higher dosage of sporozoites to challenge immunized mice, of at least 1000 spz or even higher.

This is a challenge dose that has been acceptable to reviewers in many previous papers and was originally chosen as it is routinely 100% infectious in B6 mice.16, 18-20 It is worth noting that B6 mice, compared to BALB/c mice, are highly sensitive to infection by PbA and that the iv route delivers many more spz to the liver than an intradermal mosquito bite. Thus 200 spz iv is akin to about 1000 spz intradermally. Finally, our unpublished data shows that Trm numbers do not change significantly at d13 after challenge with either 200 or 3000 spz, when mice are protected (reviewer data), suggesting that if infection is rapidly controlled then challenge with these small numbers of live sporozoites does not evoke additional boosting. We consider a 200 spz challenge highly informative of protective capacity and do not expect the results to change significantly with a 1000 spz challenge. Thus, we consider this request would be an inappropriate burden on animals that we could not justify to our animal ethics committee.


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18. Holz, L. E.; Chua, Y. C.; de Menezes, M. N.; Anderson, R. J.; Draper, S. L.; Compton, B. J.; Chan, S. T. S.; Mathew, J.; Li, J.; Kedzierski, L.; Wang, Z. F.; Beattie, L.; Enders, M. H.; Ghilas, S.; May, R.; Steiner, T. M.; Lange, J.; Fernandez-Ruiz, D.; Valencia-Hernandez, A. M.; Osmond, T. L.; Farrand, K. J.; Seneviratna, R.; Almeida, C. F.; Tullett, K. M.; Bertolino, P.; Bowen, D. G.; Cozijnsen, A.; Mollard, V.; McFadden, G. I.; Caminschi, I.; Lahoud, M. H.; Kedzierska, K.; Turner, S. J.; Godfrey, D. I.; Hermans, I. F.; Painter, G. F.; Heath, W. R., Glycolipid-peptide vaccination induces liver-resident memory CD8(+) T cells that protect against rodent malaria. Sci Immunol 2020, 5 (48).
19. Holz, L. E.; Prier, J. E.; Freestone, D.; Steiner, T. M.; English, K.; Johnson, D. N.; Mollard, V.; Cozijnsen, A.; Davey, G. M.; Godfrey, D. I.; Yui, K.; Mackay, L. K.; Lahoud, M. H.; Caminschi, I.; McFadden, G. I.; Bertolino, P.; Fernandez-Ruiz, D.; Heath, W. R., CD8(+) T Cell Activation Leads to Constitutive Formation of Liver Tissue-Resident Memory T Cells that Seed a Large and Flexible Niche in the Liver. Cell Rep 2018, 25 (1), 68-79 e4.
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28-Feb-2022

Dear Prof Compton:

Manuscript ID: CB-ART-12-2021-000251.R1
TITLE: 6’’-Modifed α-GalCer-peptide conjugate vaccines protect against liver-stage malaria

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions. In particular, both reviewers raised concerns regarding the stage of development of this approach, and therefore I also believe that considering the construct a malaria vaccine candidate is more accurate.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

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Prof Gonçalo Bernardes

Associate Editor, RSC Chemical Biology

************

Reviewer 2

All my comments have been adequately adressed by the authors

Reviewer 1

The authors have answered to all the requested points. They have added data, they have changed some sentences etc. At this stage the manuscript can be accepted for a publication on RSC ChemBiol. The work perfectly fits the aims and scope of the Journal and it will add precious input for the ChemBiol community.

I strongly wish the authors to have one day a malaria vaccine. At this stage, the authors have a very nice vaccine-candidate.

We thank the Editor and Referees for their time and comments.

The reviewers raised concerns regarding the stage of development of this approach, and consider the constructs to be more accurately described as "vaccine candidates" rather than just "vaccines"

To address this, we have changed the wording of "vaccine" to "vaccine candidate(s)" in the tile and throughout the manuscript text. A small error was also found regarding Scheme 3. Here, compound 9 should have been labelled compound 10. This error has been corrected in the manuscript text and in Scheme 3

Please find attached "marked-up" and "clean" versions of the manuscript and SI file that reflect these changes.

02-Mar-2022

Dear Prof Compton:

Manuscript ID: CB-ART-12-2021-000251.R2
TITLE: 6’’-Modifed α-GalCer-peptide conjugate vaccines protect against liver-stage malaria

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Prof Gonçalo Bernardes

Associate Editor, RSC Chemical Biology