Non-interventional Translational Study in Mild, Moderate and Severe Acne Patients

The number of total lesions, inflammatory lesions (papules and pustules), and non-inflammatory lesions (open and closed comedones) on the face will be counted. All lesions on the face should be counted, including those on the nose.

Counts of nodulocystic lesion (nodules and cysts) will be reported separately and are not to be included in the inflammatory or non-inflammatory lesion counts.

The overall severity of facial acne will be assessed using the 5-point IGA scale (Table 6) (15). Each grade is defined by a distinct and clinically relevant morphologic description that minimizes interobserver variability.

The IGA severity score assessments should be performed exclusively by site staff qualified as IGA rater. Investigators or designee should use the IGA scale provided in the study protocol throughout the study.

With the objective of evaluating the impact of different acne severities in the quality of life, participants will be asked to complete 2 quality of life (QoL) questionnaires: the Skindex-29 and the Acne-specific Quality of Life (Acne-QoL).

The Skindex-29 questionnaire is a validated health-related quality of life self-administered questionnaire to assess the impact of skin diseases on patient's quality of life (17). Skindex-29 consists of 30 items, distributed into 3 domains (symptoms, emotion and function. Each item is scored on a 5-point Likert scale, from 1 to 5. Scores are standardized to 100, with lower score indicating higher levels of QoL.

The Acne-QoL is a validated health-related quality of life self-administered questionnaire to assess quality of life among participants with facial acne (16). Acne-QoL consists of 19 items organized into 4 domains: self-perception, role-social, role-emotional and acne symptoms. Each item is scored

The investigator or designee will take digital photographs of the participants' face using Canfield Facial Image Photography Device. Photographs will be taken under standardized conditions.

Images will be captured, viewed, and uploaded using Canfield Facial Image Photography Device software which automatically checksums, encrypts, packages, and transfers the data to secure, validated and compliant web servers hosted by Canfield Clinic.

Deidentified images will be used to assess the skin condition of each participant and be the input data for the AI-based tool to derive acne severity.

Images will be analyzed with experimental AI technologies to develop and improve acne lesion count and severity grading.

Deep learning-based methods will be used to automatically detect various categories of acne lesions and assess acne severity by quantifying the number of lesions in each category. The deep learning model will be trained on the dataset with ground truth bounding box labels annotated by dermatologists, enabling it to identify lesion locations and classify each lesion type, such as inflammatory lesions (papules, pustules, nodule, and cysts), noninflammatory lesions (open comedo and closed comedo), and others (eg, scar, melasma, and nevus). Facial images for the study are captured using the Canfield Facial Image Photography Device, ensuring high-quality, standardized input for robust model performance. The trained model will output bounding boxes with lesion classifications for test images, providing a detailed and objective assessment of acne severity based on lesion c

1.Surface Binding (SB) assays using a fluorescence cytometer will be used to determine the binding efficiency of serum antibodies to the cell surface of live C. acnes bacteria. 2. Opsonophagocytic killing (OPK) assays will be used to determine the ability of antibodies to induce opsonization of C. acnes bacteria. Bound antibodies mobilize and activate immune cells against the bacterium, leading to a reduction in the bacterial cell numbers. 3. Sheep Red Blood Cells Co-Hemolysis Neutralization Assay will be used to measure serological titers of neutralizing recombinant C. acnes CAMP2 polypeptide (CAMP2)-specific antibodies. 4. Antigen-specific Antibody Titers to further explore the humoral immune response elicited against C. acnes (e.g. C. acnes types IA1 [NCTC737], IB [KPA171202]), IgG, IgA titres and IgG subclasses distribution will be measured.

• Antibody-dependent complement deposition titer documents the ability of C. acnes-specific Ig to activate the complement system via the classical pathway. For tested serum samples, the method consists of measuring the antibody-driven recruitment and deposition of the complement components C3b/iC3b on live C. acnes bacterial using flow cytometry. 6. Cell-Mediated Immunity against C. acnes antigens cytokine assessment in supernatant from whole blood culture, the cytokine responses in whole blood will be measured by either multiplex bead array assay or MSD ECL-based assay.7.Immunophenotyping of Peripheral Blood Mononuclear Cells (PBMCs) T cell profiling by flow cytometry subsequent to stimulation. PBMCs isolated from participants will be thawed and cultured with relevant reagents and then evaluated using cytometry, using BD analyzers, spectral flow and/or CyTOF as required as required to determine activated T cell signatures.

• Peripheral blood cytokine concentration will be measured in the serum of participants by either multiplex bead array assay or MSD ECL-based assay or appropriate proteomics profiling platforms such as NULISA or OLINK.

• Genomewide genetic analysis, DNA will be extracted from PBMCs isolated from the blood collected from each participant. This DNA will then be used for genetic analysis using genomewide genotyping arrays such as Infinium (Illumina).

To characterize the microbial strains, microbiome composition and transcriptional activity, skin swabs will be collected from the participants' forehead and cheeks. For sequencing assays, material from swabs will be dislodged and submerged in DNA/RNA shield and stored at -80°C prior to nucleic acid extraction. Microbial isolation will be done according to procedures described in 4.6. Analyses such as microbiome metagenomics profiling (taxonomic profiles, relative abundance, diversity, richness) and amplicon sequencing of isolates (C. acnes Single Locus Sequence Type) could be performed alongside similar analysis from follicular extracts. Alongside this metatranscriptomic analysis of collected microbial material will be undertaken. Collected samples will be processed and stored at sites according to the best practice for each material type prior to downstream analytical assays.

Skin biopsies will be obtained and stored in an appropriate media (e.g. RPMI, PBS, or formalin) by clinical staff and kept at 4°C to await transport to A*STAR. To study components of the acne microenvironment skin biopsies will be prepared and analysed following best practices and aligned with manufacture's protocols for spatial and/or single cell RNAseq. Data will be pre-processed using the supplier's pipeline. For downstream analysis, including normalization, integration, dimensionality reduction, annotation and detection of differentially expressed genes, the resulting matrices will be processed using R or Python packages such Seurat or Monocle or by vendor specific programs.

Skin surface material assessment such as metabolomics or proteomics will be performed from skin tape strips to detect small molecules and larger lipids including ceramides, fatty acids, and triglycerides or skin surface protein expression. Skin tapes material will be extracted with organic solvents and injected into dual-column liquid chromatography-triple quadrupole mass spectrometry for high-throughput metabolomics measurements. Similarly, skin tape material could be extracted for proteomics analysis prior to LC-MS/MS analysis. Multivariate analyses will be conducted to differentiate participant group clustering and identification of top metabolites or proteins driving these differences.

Isolation of microbes from the skin surface and/or the follicular ostia will be performed with automation and/or standard selective culture and plating techniques. We will use conditions to mainly target C. acnes strains as the species of particular interest. Other strains such as Staphylococcus spp. may also grow under similar conditions that will also be isolated and banked for study. Representative isolates will be genome sequenced (Illumina) and genome assembled for phylotyping and gene analysis such as for virulence/vaccine target.