Establishment of a Dynamic Ear Inflammation Model in Rats for Acne Vulgaris and Evaluation of Adjuvanted Inactivated Cutibacterium acnes-Based Vaccines Efficacy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although the manuscript "Establishment of a Dynamic Auricular Inflammation Model in Rats for Acne Vulgaris and Evaluation of Adjuvanted Inactivated Cutibacterium acnes-Based Vaccines Efficacy" presents a certain scientific interest, there are some important comments:

1. Although the sample size (n=5-6 per group/time point) appears reasonable, it might be small enough to impair statistical power, and a post-hoc analysis should be included to ensure it has sufficient power for detecting differences.
2. Methods of randomization and blinding in allocating animals and in measuring outcomes, for instance, ear thickness and histopathology scores, are not described in detail, and how groups are randomized, for instance, through stratified block randomization, and whether blinded assessments are done, among others, should be described.
3. The heat inactivation method of HI-C. acnes at 56°C for 30 min is quite simple but could be damaging to heat-labile antigens; it must be shown that antigenic identity has been maintained after inactivation and that other techniques, such as fixation in formalin, are available for comparison.
4. Adjuvants WS03 and MA107b are added without adequate characterization; a short description or bibliographic note regarding their immunological characteristics is required in the context of selection.
5. The volume of the intradermal injection (50 µL) and the bacterial number (8x10^9 CFU/mL) appear to be excessive for the ear size and promote tissue necrosis rather than inflammation; add experimental data to support the bacterial concentration used.
6. Cytokine analysis employs a 13-plex analysis but records only three markers (IL-6, IL-1β, MCP-1); the analysis should include all relevant analytes or rationalize the selective inclusion.
7. Statistical inferences are made solely based on Mann-Whitney U tests, without any adjustment for multiple comparisons; for instance, over time points or cytokines, and Bonferroni or FDR adjustments could be made for any Type I error.
8. The finding that vaccine-induced protection can be regarded as “strongly correlated with humoral immunity” (Abstract, Discussion), without considering the possible role of the cellular response, can be explored in greater detail by correlating the data (Spearman rank correlation between IgG levels and outcomes) or commenting on T-cell immunity (IFN-γ Elispot).
9. Scoring histopathology (Table 1) is semi-quantifiable but does not include criteria for inter-rater reliability; to add to its objectivity, values for Cohen’s K or ICC should be included.
10. Temporal dynamics are adequately described; however, there is a lack of multivariate analysis (mixed effects ANOVA) that could incorporate variables such as bacterial load, cytokines, and ear thickness; these should be added.
11. WS03 or MA107b vaccine efficacy comparisons exhibit a trend (e.g., superior protection from MA107b), but there is no test for interaction; add analyses to clarify differences for each adjuvant.
12. There are some typographical and grammatical errors. Please fix it.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper describes an inflammatory model produced by the injection of a high dose of live Cutibacterium acnes into rat ears and describes experiments that show that vaccination with a Cutibacterium acnes reduces the inflammatory response and enhances clearance or killing of live bacteria.

As with other papers using the rat and mouse models there is no evidence that the Cutibacterium acnes grow in the skin and inflammatory responses have been observed with killed bacteria. My calculation shows that 4x10^8 bacteria were injected and on day one there were less than 1.5 x10^8 in the ear.  The word proliferate should be removed from line 226 unless there is evidence for proliferation. Perhaps a 12 hour time point might help.

The method of preparing the tissue homogenates used for the cytokine measurements should be described.

The conditions of baking and peroxidase blocking for the IHC could be given.

The constitution of the WS03 and  MA107b should be given. Neither of these designations were found on a Medline search.

The vaccination studies are interesting but the injection of bacterial extracts can induce suppressive effects. The paper would be improved if the effect of the vaccination on another inflammatory response was examined.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf