Dear Professor Gale Jr.,

Thank you very much for submitting your manuscript "Sustained IL-15 response signature predicts RhCMV/SIV vaccine efficacy" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by two independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Guido Silvestri

Associate Editor

PLOS Pathogens

Thomas Hope

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Although a novel RhCMV/SIV vaccine (68.1) has been demonstrated to provide virologic control and ultimately clearance of SIV infection in approximately half of vaccinated rhesus macaques, mechanisms that underlie this protection are not understood. This is an issue of great importance to the field as the correlates of immune protection from SIV and HIV are not understood. To attempt to provide mechanistic insight into these observations, transcriptomic analyses were performed in two cohorts of rhesus macaques who received the vaccine (n=30 and n=15) as well as five rhesus macaques who were treated with an IL-15 superagonist. The large number of animals studied is a strength of the study. This approach to understand mechanisms underlying protection conferred by the RhCMV/SIV vaccine is ambitious, but well reasoned.

Global transcriptomic profiling of mRNA in whole blood samples was performed at multiple time points and analyzed by principal component analysis and determination of differential expression of genes following vaccination. Differentially induced genes occurred rapidly after the first vaccine (day 1), were variably reduced, rebounded after the booster vaccine and were generally higher after the booster in the protected macaques compared to unprotected ones. Differences in this signature persisted and existed at the time of challenge. Further analysis of genes that were differentially expressed between protected and nonprotected animals revealed 2,272 genes that fell into four major clusters identified by clustering analysis. Vaccine induced gene clusters were associated with innate immune pathways including TLR signaling, cytokine response pathways, inflammasome/cell death signaling, and T cell/TCR signaling genes. IL-15 expression was enriched in the protected animals and was identified as a significant upstream regulator of the vaccine protection signature. Further evidence that the signature transcriptome was heavily influenced by IL-15 came from transcriptomic analyses in the animals treated with the IL-15 superagonist, who demonstrated at day 1 multiple transcriptome clusters that overlapped with those seen in the transcriptome signature associated with vaccine protection. These findings strengthen the conclusion that IL-15 is associated with many of the signature changes.

The whole blood gene signature was next validated using a different cohort of RM that had been vaccinated with both the 68-1 RhCMV/SIV vaccine and a non-protective variant (68-1.2). Six of the 15 vaccinated rhesus macaques exhibited the protected phenotype. Vaccine responses in both the original cohort and the validation cohort reportedly showed a higher correlation with the IL-15 response than in non-protected animals. Nevertheless, the data shown in Figure 5 are not compelling, and there is no statistical analysis associated with these results.

The authors conclude that the IL-15 response signature is a predictor of vaccine response that is linked to protection. They postulate that “tuning” by persistent induction of IL-15 and associated immunoregulatory pathways may be required for the MHC-E restricted SIV-specific CD8+ T cells to mediate arrest of viral replication. Because cells become refractory to IL-15 after continuous stimulation, they speculate that low, persistent IL-15 production is essential. They acknowledge that the basis for differential induction and maintenance of the protective signature among rhesus macaque is not clear, nor is the mechanism that accounts for RhCMV/SIV vectors to induce this response that is not induced by other vaccines.

The conclusions of the authors that the IL-15 response signature is a predictor of vaccine response is somewhat supported by the data. Nevertheless, it is not clear how much overlap there is between protected and unprotected animals. Can protection be predicted prior to challenge? The findings that are presented certainly suggest a potential role for IL-15. This information is interesting to the field, since IL-15 is under intensive investigation in several HIV cure protocols. Nevertheless, the studies presented in the manuscript are essentially descriptive, and not definitive. More compelling evidence that IL-15 is a key mediator of the vaccine-induced protection could come from attempts to modify the IL-15 response at the time of vaccination or challenge.

Reviewer #2: This study by Barrenas and colleagues investigates the mechanism of vaccine elicited protection from mucosal SIV challenge using the Rh-CMV vaccine in rhesus macaques. The RhCMV vaccine has been one of the most promising vaccine candidates in recent years, and a series of high impact papers have demonstrated its reproducibility and rigour in affording protection at slightly over 50% of challenged. In a set of follow-up studies, partial explanation of the protection has been attributed to the ability of the RhCMV vector to elicit long-standing mucosal SIV-specific effector memory CD8+ T cells, and to engender noncanonical restriction of CD8 T cells to recognize SIV antigens via MHC class II and class E presentation. In the current study, which has been highly anticipated, the authors employ high-throughput transcriptomics to query longitudinal gene expression after RhCMV vaccination. The goal was to identify additional pathways influenced by RhCMV 68-1 vaccine-elicited protection.

The study is comprised of a massive amount of RNA-Seq from a set of frequent longitudinal blood samples. The study is comprised of an impressive amount of complex biofinformatics, which required significant unorthodox statistical methodology. The study is very well-powered, and uses a very innovative study design: they leverage the well-powered nature of the parent NHP studies (n = 15 total) and the sample sizes of the subgroups of animals in matched sets of “protected” and “unproctected” with each group consisting of n = 7-9, more than adequate for robust power in a blood-based transcriptomics study expected effect sizes by most vaccines. The primary finding of the study is that animals exhibiting protection have a consistent and strong induction of IL15 signalling pathways. Finally – the study uses an exceptionally extensive orthologous validation of the IL15 gene signatures to support their model. Some of the weaknesses are 1. Given that extensive prior work has established significant portion of the mechanism by which this vaccine affords protection (non-conventional MHC restriction) – the impact of the current study is lessened 2. The current conclusively associates a gene signature with protection, but offers little insight into how this signature mechanistically acts. However, these concerns are mitigated by the clear importance of the scientific problem (understanding all aspects how this promising vaccine platform works). Lastly- in my opinion, this study is an example of an effective transcriptomics study – it has provided a high confidence hypothesis to explain a complex biomedical question, i.e. the association of IL15 signaling to RhCMV-mediated protection- and while further validation (i.e. targeting IL15) will be critical to the advancement of this model – they are beyond the scope of the current study.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. Experimental evidence that alteration of the IL-15 response (augmentation or ablation) either at the time of vaccination or challenge changes vaccine protectiveness would provide more compelling evidence that IL-15 is central to vaccine protection.

2. Identification of definitive parameters that distinguish protected versus unprotected animals prior to challenge would strengthen the argument that a true signature exists.

Reviewer #2: 1. The method in which the data presented in Figure 2D is somewhat geared towards presenting coherent heatmaps: by using genes in PC1 together they will naturally act the same way. While its fine to leave this figure – I would suggest augmenting Figure 2 with a more straightforward traditional analysis: (i) provide Boolean diagrams of the overlaps of DEGs between the Oral and SubQ protected gene expression, and also with the unprotected. (ii) Include a heatmap in which the top ~1000-2000 DEGs across all comparisons are clustered – to show natural coherence of the genes (i.e. without the filtering effect of the PCs).

2. Figures 2A and 2B can be omitted, they don’t add much to the story, and given the way they are calculated, are not really insightful. Alternatively, cluster and/or heatmaps of correlation co-efficients for post-vaccine timepoints should be shown for comparison.

3. The rhesus annotation MMul_1 – is not widely used… do they mean Mmul_10? In any case, it would not be practical to redo the analysis, but further description of this annotation in terms of #’s of genes would be helpful in the Methods.

4. The numbers of DEGs at some time-points seems abnormally high… according to the methods, the reference yielded ~12,000 transcripts for analysis, and the Oral Protected group had >2000 genes upregulated and >2000 genes downregulated for a total of >4000 genes of 12,000. Further explanation is warranted.

5. The Discussion is underdeveloped – likely from formatting the length for other journals. More in depth discussion of the caveats of the study, and potential mechanisms of IL15 beyond “non-conventional T cell responses” would be helpful. What about NK cells? Does IL15 enhance mucosal T cell responses.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: 1. Some minor typos, and potential corrections: is it PAXgene miRNA or RNA kits? In the Abstract “toll-lie”.

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Reviewer #2: No

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Dear Professor Gale Jr.,

We are pleased to inform you that your manuscript 'Interleukin-15 response signature predicts RhCMV/SIV vaccine efficacy' has been provisionally accepted for publication in PLOS Pathogens.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Guido Silvestri

Associate Editor

PLOS Pathogens

Thomas Hope

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):