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PONE-D-26-18832

Adjuvants MPLA and SMNP induce antiviral immunity and indirectly revert HIV-1 latency

PLOS One

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Additional Editor Comments :

The authors need to address the reviewers specific comments especially around statistical analysis and data interpretation

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Reviewers' comments:

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: I Don't Know

Reviewer #2: No

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: This manuscripts evaluates the effects of the vaccine adjuvants MPLA and SMNP on immune activation and inducible HIV reservoir in people with HIV on suppressive ART. SMNP is a new adjuvant that has been evaluated in vaccine studies in healthy volunteers and will also be part of therapeutic vaccine studies. The authors performed as series of in vitro studies and showed that both adjuvants activated dendritic cells through TLR4 signaling and induced pro-inflammatory cytokine production. SMNP was superior to MPLA. Interestingly, SMNP, but not MPLA, reduced the size of the inducible HIV reservoir ex vivo without detectable bystander cytotoxicity, likely through cytokine-mediated latency reversal and immune activation. These findings support further investigation of SMNP as a potential adjuvant for therapeutic HIV vaccination strategies. The data is presented clearly and it relevant to the vaccine field and HIV in particular as several ongoing and planned studies will use SMNP as the adjuvant.

Studies in healthy volunteers and pre-clinical studies shows that SMNP is a strong adjuvant that improves immunogenicity of different protein vaccines. The data of PWH is also reassuring that despite immune dysregulation activation of dendritic cells occur. HIV-specific immune responses were not directly evaluated in this study. The ex vivo data presented show that SMNP-mediated immune activation and cytokine-mediated latency reversal are not associated with cytotoxicity. However it’s impact on HIV-specific immune responses was not evaluated. It would strengthen the paper to include such evaluations, if possible.

Since effects on the HIV reservoir would be of interest in the context of therapeutic vaccination, the following statement in the abstract “However, as reservoir reduction was observed ex vivo, further evaluation is warranted to ensure safe use of such adjuvants in PWH” in unclear. It has been shown in studies of vaccination or co-infection that while viral blips may occur, during ART viral suppression is not compromised. The authors should clarify the statement about safety in the context of HIV and the broader goals of remission strategies.

Reviewer #2: The manuscript addresses whether MPLA and SMNP adjuvants induce innate immune activation in ART-suppressed PWH and if this lowers the inducible HIV-1 reservoir ex vivo. The conclusion that SMNP is more immunostimulatory than MPLA is supported by the activation and cytokine data and the observation that SMNP reduces inducible reservoir ex vivo has potential. The authors appropriately acknowledge that these are ex vivo findings requiring further evaluation before clinical translation. I ask that they also consider the following comments.

Major Comments

1. The statistical analysis section is too vague and not the best approach for the experimental design. The authors state that two-way ANOVA, one-way ANOVA, Brown-Forsythe/Welch ANOVA, and post-hoc corrections were used, but they do not clearly specify which test was used for each figure, whether donor-matched repeated-measures analyses were used, what correction was applied for each. Since many comparisons involve repeated measures across doses or treatments within the same donors, paired repeated-measures models or mixed-effects models would be more appropriate than simple ANOVA. Exact p values should be reported where possible and figure legends should clearly state the statistical test, n, and correction method for each panel.

2. I recommend more conservative language around reservoir reduction. The data suggest reduced inducible virus after SMNP exposure, but the mechanism is inferred rather than directly shown. The authors should avoid implying selective elimination of infected cells unless supported by additional evidence. Alternative explanations, including altered inducibility, assay interference, cytokine-mediated effects on outgrowth/readout, or changes in target-cell activation should be more explicitly discussed.

3. The manuscript language is clear but would benefit from copy editing. There are multiple grammatical errors, spacing/formatting issues, and imprecise phrasing, including “ex vivowithout,” “supressed,” “monoplo,” and several awkward sentences in the Introduction and Discussion. These do not prevent interpretation but should be corrected.

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Reviewer #1: Yes: Marina Caskey

Reviewer #2: No

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Adjuvants MPLA and SMNP induce antiviral immunity and indirectly revert HIV-1 latency

PONE-D-26-18832R1

Dear Dr. Kootstra

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Alan Landay

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: I Don't Know

Reviewer #2: Yes

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-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: The authors have addressed my comments appropriately. I would recommend softening the statement in the abstract about a substantial reduction in the reservoir size. While the differences are statistically significant, these are ex vivo assays and it is far from clear that this level of reduction would have meaningful consequences in vivo. It is an interesting finding and of interest to the HIV remission field, however the statement could be modified to something like "reduced the size of the inducible reservoir ex vivo".

Reviewer #2: The authors have adequately addressed the concerns raised during the initial review. I recommend acceptance pending only minor editorial revisions.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Marina Caskey

Reviewer #2: Yes: Joshua Cyktor

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