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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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3. 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: No

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: In this paper, Qabar CM et. al. report the generation of a recombinant M. bovis BCG strain that overexpresses an isoprenoid metabolite and phospho-antigen known as HMBPP. HMBPP is a stimulus which expands Vγ9Vδ2 T-cell subsets in the host, and these same cells are believed to play an important role in containing TB disease. The authors postulate that an rBCG strain of this nature would be a more potent TB vaccine than standard BCG.

This concept is innovative and novel. This paper reports a preliminary, partial dataset to support their hypothesis.

Concerns

1. Fig. 3c. There is insufficient evidence to claim that overexpression of GcpE induces Vg9Vd2 cell expansion. The claim is based on Fig. 3c where we are shown on 2 determinations (BCG-WT) compared to 3 determinations (BCG+gcpE-OE). This sample size is too low. In contrast, Fig. 2c (5 dilutions tested on multiple replicates) is convincing evidence that BCG+MEPsynth strain does not elicit the intended Vγ9Vδ2 response. However, we are not shown the same 5 dilutions tested on multiple replicates for the BCG+gcpE-OE strain. The authors should either show more data for the GcpE strain or withdraw the claim that the GcpE overexpressor induces Vg9Vd2 cell expansion.

2. Fig 3c. Why are we only shown the results from Donor E? The legend indicates there was a second donor tested.

3. Since the synthetic biology approach did not increase the expansion of Vγ9Vδ2 T-cells, the authors should consider modifying the title of the manuscript

4. In addition to measuring Vg9Vd2 cell expansion for the recombinant BCGs, it would be valuable to know what the actual levels of HMBPP are.

5. Why were only BCG lysates tested in the Vγ9Vδ2 T-cell expansion assay and not the live BCG strains?

6. Figure 1 has several inconsistencies which should at the very least be discussed as limitations: (i) Why do the mutants show a growth defect in the absence of ATc-mediated knockdown. (ii) Why does the “No guide control” exhibit a growth defect in presence of ATc. (iii) While the spotting assay is a quick and dirty approach, to claim that a gene is essential, a CFU-based growth kinetic analysis should be done. (iv) Lastly, qRT-PCR validation is necessary to show that the knockdowns are functioning correctly.

7. Fig 2b and 3b. Why are mRNA levels so low with these strong promoter constructs at 0.00015 (Fig. 2b) or 0.002 (Fig. 3b) relative to 16S rRNA?

8. Fig. 2c and 2c should have statistics included.

9. Inclusion of the flow cytometry dot plots and gating strategy for Fig. 2c and 3c would be helpful.

10. Line 58-59: The statement that Vγ9+Vδ2+ γδ T cells increase to ~50% of total blood T cells should be qualified. Other studies show lower values closer to 20% (eg Kroca et al. PMID: 10792377)

11. Lines 207-210. Why do the authors think that dxr and ispH appear to be essential in BCG by CRISPRi in their hands, but that in Mtb transposon mutants were viable as reported by the DG Russell lab (ref 63).

12. Line 220. The text states the essential gene rpoB was targeted but Fig 1b shows folC was targeted. Please clarify.

13. Line 329. Discussion should be toned down. Feedback inhibition was a possible reason, but this was not formally demonstrated.

14. The approach for increasing HMBPP used herein was essentially “make more” or “turn up the faucet”. In the discussion, it might be worth mentioning that another way to increase HMBPP levels in BCG would be to conditionally knockdown ispH and thereby reduce usage or “close the drain”

Reviewer #2: The manuscript presents a novel synthetic biology-based strategy to enhance TB vaccine efficacy by engineering the MEP pathway in BCG to increase HMBPP production, thereby stimulating Vγ9Vδ2 T cells. This concept is timely and innovative, combining metabolic engineering with immune modulation to address current limitations in TB vaccination strategies. The rationale and design are well-justified, and the use of human PBMCs enhances translational relevance. However, several critical revisions are required to ensure methodological rigor, data transparency, and appropriate interpretation:

A. Methodological Transparency: Clearly indicate sources of strains, plasmids, antibodies, and include gene identifiers such as Rv numbers or BCG locus tags.

B. CRISPRi Knockdown Assay – Figure 1B

· The spot assay lacks proper controls and normalization:

o Include a dCas9-only or scrambled sgRNA control.

o Ensure equal plating, incubation timing, and inoculum across plates. The no-guide control shows reduced growth upon ATc induction, indicating possible non-specific effects of dCas9 or ATc, which compromises interpretation of gene-specific knockdowns. Additionally, unequal bacterial input and apparent differences in incubation timing between plates further confound comparison. The absence of quantitative measurements (e.g., CFU counts or spot intensity) and replicates limits the robustness of the conclusions.

o Provide CFU counts or quantification of spot intensity across replicates.

· Without these controls, gene essentiality interpretations are weakened.

C. Gene Expression Analysis – Figures 2B and 3B

· The RT-qPCR data should be reanalyzed and presented using the ΔΔCt method.

o Display data as log₂ fold changes with appropriate statistical tests and annotations.

o This will improve clarity, comparability, and rigor.

D. PBMC Stimulation – Figures 2C and 2D

· Normalization of Lysates: OD₆₀₀-based normalization is insufficient:

o Include growth curves or CFU counts to demonstrate equal biomass.

o Specify how lysate input was standardized.

o Authors are requested to quantify HMBPP levels via LC-MS or other methods to support biological interpretation.

· Statistical Annotation (Figure 2D):

o Please include statistical comparisons between:

§ HMBPP vs. BCG WT

§ HMBPP vs. BCG+MEP^synth

o Add p-values and update the legend accordingly.

o Clarify the type and number of replicates used (biological vs. technical) for each experiment.

In particular, for the PBMC-based assays, the number of data points per group appears inconsistent across conditions—for example, in Figure 2D. If the assays were performed using PBMCs derived from the same set of human donors and stimulated simultaneously with different conditions, the number of samples (i.e., data points or “dots” per group) should be identical across all treatment groups. Please clarify the experimental setup and provide an explanation for these discrepancies in sample numbers.

E. Biosafety and Ethical Approvals

· The manuscript must clearly state Institutional Biosafety and IRB approvals for:

o Genetic manipulation.

o Use of human PBMCs.

· Include protocol numbers or justification for exemption, if applicable.

F. Statistical Analysis

· Indicate:

o Statistical tests used in each figure.

o Exact p-values or significance thresholds.

o Confidence intervals where applicable.

G. Discussion

· Expand the discussion on potential limitations, possibility of feedback regulation in MEP pathway, how isoprenoid over-production alters the production of classical antigens of BCG.

· Discuss absence of protective efficacy data. Reframe claims of protective immunity as speculative, pending validation in appropriate in vivo models.

· If you are adding HMBPP quantification, please discuss that.

· Strengthen context with references to:

Clinical efforts involving Vγ9Vδ2 T cell-targeted vaccines.

o Metabolic engineering strategies in mycobacteria.

I. Compliance with Journal Requirements

· Include detailed author contribution statements as per PLOS ONE guidelines.

Overall, the manuscript holds promise but requires substantial revision. Addressing these issues will significantly enhance the robustness and impact of the study.

Reviewer #3: The manuscript presents an innovative synthetic biology approach to enhance BCG-mediated expansion of Vγ9Vδ2 T cells by engineering the isoprenoid biosynthesis pathway in BCG, specifically to overproduce HMBPP. The underlying hypothesis is sound and the objective highly relevant in the context of TB vaccine enhancement strategies, particularly considering the unique role of Vγ9Vδ2 T cells in primate immunity. However, in its current form, the study appears somewhat inconclusive and requires additional data to substantiate its conclusions.

Major Concerns:

1) A critical shortcoming of the study is the lack of direct quantification of HMBPP in the engineered BCG strains. Although gene expression levels of MEP pathway genes and gcpE are provided, they cannot serve as a surrogate for actual metabolite levels. Without intracellular or extracellular quantification of HMBPP, the interpretation that gcpE overexpression leads to increased HMBPP accumulation remains speculative. It is imperative that the authors include HMBPP quantification (e.g., via LC-MS/MS or other validated methods) in both synthetic operon-expressing and gcpE-overexpressing strains to substantiate their claims.

2) In Figures 2C, 2D, and 3C, the number of biological replicates (i.e., PBMC donors) and technical replicates is either unclear or inconsistently described in the legend. This limits the interpretability and statistical robustness of the expansion assay data. Authors should clearly state the number of independent donors, replicate wells, and how variability was accounted for in the analyses (mean ± SD, statistical tests, etc.). A separate sub-section on ‘Statistical Analysis’ should be added to ‘Materials and Methods’ section.

3) While expansion of Vγ9Vδ2 T cells is demonstrated, no functional immune response (e.g., cytokine release, or cytotoxic activity) is assessed to confirm that these expanded cells are immunologically competent. Inclusion of such functional data (IFNγ, Granzyme B, etc.) would strengthen the manuscript and its relevance to vaccine efficacy.

Minor Comments:

1) Reference [29] appears to have a non-standard font or formatting. This should be corrected.

2) The manuscript frequently repeats the background on Vγ9Vδ2 T cells and HMBPP throughout the introduction and results. This dilutes the emphasis on what was experimentally discovered. The authors should consider condensing these sections to improve clarity and focus.

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Reviewer #1: No

Reviewer #2: Yes: Ruchi Jain DeyRuchi Jain Dey

Reviewer #3: No

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Dear Dr. Cox,

plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Atul Vashist, PhD

Academic Editor

PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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 #3: Yes

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: All concerns adequately addressed. Crazy that I have to write 100 characters in order for the website to accept this response

Reviewer #3: The revised manuscript shows clear improvement and the work is progressing in a strong direction. One specific point still requires clarification to strengthen the reproducibility of your key findings:

PBMC Expansion Assay Methodology

The PBMC stimulation workflow is appropriate; however, the description of bacterial lysate preparation would benefit from quantitative detail. At present, lysate input is reported only as dilution factors, without specifying the actual concentration of the stimulatory material. Since several processing steps (bead-beating, lysate recovery, 3 kDa filtration, volume adjustments) can alter the final composition, Bacterial culture OD normalization alone may not ensure equivalent stimulant load between samples.

Because the PBMC expansion data (e.g., Fig. 2C, 3C, S2 B–D) are central to the manuscript’s conclusions regarding the differential effects of engineered rBCG versus wild-type BCG, it would substantially strengthen the study to normalize lysates to an absolute metric- such as total protein concentration (μg/mL or μg/well, measured by BCA/Bradford) or a comparable mass-based standard.

For clarity and rigor, it is recommended to include:

Quantification of lysate protein concentration prior to use (μg/mL).

Normalization of all lysates to the same protein input per well during stimulation.

This addition will enhance comparability across experimental arms and ensure that observed differences in PBMC expansion reflect biological effects rather than variation in lysate load. Addressing this point will more firmly support the manuscript’s core conclusions.

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what does this mean? ). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #3: No

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Leveraging a synthetic biology approach to enhance BCG-mediated expansion of Vγ9Vδ2 T cells

PONE-D-25-24963R2

Dear Dr. Cox,

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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PLOS One

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Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

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

Reviewer #3: Yes

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

Reviewer #3: 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 #3: Yes

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

Reviewer #3: Yes

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Reviewer #3: In the revised manuscript, all concerns adequately addressed. The study represents a step forward in the effort to develop a better vaccine for TB.

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Reviewer #3: No

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