Transfer of modified gut viromes improves symptoms associated with metabolic syndrome in obese male mice

Metabolic syndrome encompasses amongst other conditions like obesity and type-2 diabetes and is associated with gut microbiome (GM) dysbiosis. Fecal microbiota transplantation (FMT) has been explored to treat metabolic syndrome by restoring the GM; however, concerns on accidentally transferring pathogenic microbes remain. As a safer alternative, fecal virome transplantation (FVT, sterile-filtrated feces) has the advantage over FMT in that mainly bacteriophages are transferred. FVT from lean male donors have shown promise in alleviating the metabolic effects of high-fat diet in a preclinical mouse study. However, FVT still carries the risk of eukaryotic viral infections. To address this, recently developed methods are applied for removing or inactivating eukaryotic viruses in the viral component of FVT. Modified FVTs are compared with unmodified FVT and saline in a diet-induced obesity model on male C57BL/6 N mice. Contrasted with obese control, mice administered a modified FVT (nearly depleted for eukaryotic viruses) exhibits enhanced blood glucose clearance but not weight loss. The unmodified FVT improves liver pathology and reduces the proportions of immune cells in the adipose tissue with a non-uniform response. GM analysis suggests that bacteriophage-mediated GM modulation influences outcomes. Optimizing these approaches could lead to the development of safe bacteriophage-based therapies targeting metabolic syndrome through GM restoration.

Page 1 of 19 Fig. S1: Overview of mouse phenotypic characteristics after being treated with the different FVTs.A) Oral glucose tolerance tests (OGTT) measured at study week 13 (18 weeks old) (error bars describe mean±SD) B) Total area under the curve (tAUC), and C) to E) sub-categories of MAFLD activity score evaluating hepatocyte injury, inflammation, and steatosis at study week 18 (23 weeks old).The labels of *, **, *** between the FVT treatments and obese control mice represent adjusted p < 0.05, 0.01, 0.001 (two-side Wilcoxon rank-sum test with FDR correction); the unadjusted p is presented in the figure when it is less than 0.05.The red star on top of the boxplot of the treatment represents a p < 0.05 between the treatment and the lean control mice (two-side Wilcoxon rank-sum test).Combined boxplots distributions include median, min, max, 25 and 75 percentiles, and outliers (more than 1.5 IQR).Combined boxplots distributions include median, min, max, 25 and 75 percentiles, and outliers (more than 1.5 IQR).Combined boxplots distributions include median, min, max, 25 and 75 percentiles, and outliers (more than 1.5 IQR).Combined boxplots distributions include median, min, max, 25 and 75 percentiles, and outliers (more than 1.5 IQR).Page 13 of 19

Fig. S2 :
Fig. S2: Proportions of immune cells in the mesenteric lymph node (MLN) at study termination (23 weeks old).A) to L) Show the overall flow cytometric profile in the MLN tissue of the different treatments.The labels of *, **, *** between the FVT treatments and obese control mice represent adjusted p < 0.05, 0.01, 0.001 (two-side Wilcoxon rank-sum test with FDR correction), the unadjusted p is presented in figure when it is less than 0.05.The red star on top of the boxplot of the treatment represents a p < 0.05 between the treatment and the lean control mice (two-side Wilcoxon rank-sum test).

Fig. S3 :
Fig. S3: Cytokine profiles of blood serum sampled at termination (23 weeks old).A) to J) are showing the overall cytokine profile in the mouse blood serum of mice treated with FVT-ChP, unmodified FVT, and the two dietary control groups.The labels of *, **, *** between the FVT treatments and obese control mice represent adjusted p < 0.05, 0.01, 0.001 (two-side Wilcoxon rank-sum test with FDR correction), the unadjusted p is presented in figure when it is less than 0.05.The red star on top of the boxplot of the treatment represents a p < 0.05 between the treatment and the lean control mice (two-side Wilcoxon rank-sum test).Combined boxplots distributions include median, min, max, 25 and 75 percentiles, and outliers (more than 1.5 IQR).

Fig. S4 :
Fig. S4: Relative bacterial abundance at termination (23 weeks old).A) -H) Boxplots showing the relative abundance of selected bacterial taxa.I) Boxplots of the ratio between Bacteroidetes:Firmicutes that were observed with the different treatment groups.J) Bar plot of phyla distribution based on treatment group.The labels of *, **, *** between the FVT treatments and obese control mice represent adjusted p < 0.05, 0.01, 0.001 (two-side Wilcoxon rank-sum test with FDR correction), the unadjusted p is presented in figure when it is less than 0.05.The red star on top of the boxplot of the treatment represents a p < 0.05 between the treatment and the lean control mice (two-side Wilcoxon rank-sum test).

Fig. S7 :
Fig. S7: Proportions of immune cells in adipose tissue of the unmodified FVT treatment stratification analysis at study termination (23 weeks old).A) to L) are showing the overall flow cytometric profile in the mouse adipose tissue.The labels of *, **, *** between the FVT treatments and obese control mice represent adjusted p < 0.05, 0.01, 0.001 (twoside Wilcoxon rank-sum test with FDR correction), the unadjusted p is presented in figure when it is less than 0.05.The red star on top of the boxplot of the treatment represents a p < 0.05 between the treatment and the lean control mice (twoside Wilcoxon rank-sum test).Combined boxplots distributions include median, min, max, 25 and 75 percentiles, and outliers (more than 1.5 IQR).

Fig. S8 :
Fig. S8: Proportions of immune cells in mesenteric lymph node (MLN) of the unmodified FVT treatment stratification analysis at study termination (23 weeks old).A) to L) shows the overall flow cytometric profile in the MLN tissue of the different treatments.The labels of *, **, *** between the FVT treatments and obese control mice represent adjusted p < 0.05, 0.01, 0.001 (two-side Wilcoxon rank-sum test with FDR correction), the unadjusted p is presented in figure when it is less than 0.05.The red star on top of the boxplot of the treatment represents a p < 0.05 between the treatment and the lean control mice (two-side Wilcoxon rank-sum test).Combined boxplots distributions include median, min, max, 25 and

Fig. S10 :
Fig.S10: Overview of the applied FVT viromes.A) -D) Epifluorescence microscopy images of the different applied FVT viromes (before being diluted to similar VLP/mL concentrations) that were stained with SYBR Gold to count VLP/mL (the scale bar is 1 µm).E) Viral taxonomy composition bar plot of the four applied FVTs on the family level including the relative abundance in percentages of eukaryotic viruses in each FVT treatment.

Table S1 :
Overview of the oral glucose tolerance (OGTT) statistics at each time point at study week 13 (18 weeks old).
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Table S3 :
List showing the included bacterial and phage strains, relevant strain-specific information, and growth conditions.

Table S4 :
Overview of cage-associated effects of phenotypical factors statistics.Comparison was done with Wilcoxon