Abstract
The ubiquitous skin colonist Staphylococcus epidermidis elicits a CD8+ T cell response pre-emptively, in the absence of an infection1. However, the scope and purpose of this anticommensal immune programme are not well defined, limiting our ability to harness it therapeutically. Here, we show that this colonist also induces a potent, durable and specific antibody response that is conserved in humans and non-human primates. A series of S. epidermidis cell-wall mutants revealed that the cell surface protein Aap is a predominant target. By colonizing mice with a strain of S. epidermidis in which the parallel β-helix ___domain of Aap is replaced by tetanus toxin fragment C, we elicit a potent neutralizing antibody response that protects mice against a lethal challenge. A similar strain of S. epidermidis expressing an Aap-SpyCatcher chimera can be conjugated with recombinant immunogens; the resulting labelled commensal elicits high antibody titres under conditions of physiologic colonization, including a robust IgA response in the nasal and pulmonary mucosa. Thus, immunity to a common skin colonist involves a coordinated T and B cell response, the latter of which can be redirected against pathogens as a new form of topical vaccination.
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Data availability
Source data are provided for every graph shown in this study in source data files associated with each figure and three replicates of uncropped immunoblots are shown in Supplementary Figs. 1–3. New S. epidermidis genomes can be downloaded on NCBI: SAMN17729819, SAMN17729840, SAMN17729842, SAMN31818776, SAMN31819003, SAMN35843294, SAMN44625711-SAMN44625714 and at https://doi.org/10.5281/zenodo.14183493 (ref. 77). Source data are provided with this paper.
Code availability
All the code associated with this work can be found at https://doi.org/10.5281/zenodo.14183493 (ref. 77).
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Acknowledgements
We are deeply indebted to members of the Fischbach laboratory for helpful discussions and suggestions, especially B. Caliando for cloning support, J. Bunker for feedback on the manuscript, A. Cheng for microbiology input, N. Johns for support with S. epidermidis sequencing and assembly; members of the Victora laboratory, especially G. Victora, A. Schiepers and L. Mesin for sharing protocols and insightful conversations; A. Bilate for providing feedback and helpful discussions; M. Prado, A. Espinoza and J. Au for keeping the laboratory running; J. Merriman for coordinating with Stanford Microbiome Therapies Initiative; K. Lemon and B. Moore for sharing strains; M. Pirazzini and C. Montecucco for assistance with the tetanus toxin challenge experiments; members of the Stanford University Veterinary Service Center for animal husbandry; the electron microscopy facility of Washington University, especially G. Strout for guidance with electron microscopy sample preparation, and staff members of the Stanford University shared FACS facility for assistance with flow cytometry analysis (National Institutes of Health (NIH) grant no. 1S10OD026831-01). The computational resources of the NIH High-Performance Computation Biowulf Cluster (http://hpc.nih.gov) were used for this study. This work was supported by the Bill and Melinda Gates Foundation (M.A.F.), Open Philanthropy (M.A.F.), the HS Chau Foundation (M.A.F.), the Stanford Microbiome Therapies Initiative, the Swiss National Science Foundation (D.B., Early Postdoc.Mobility and Postdoc.Mobility), NIH grant nos. 5R01AI175642-02 (M.A.F.), 1K99AI180358-01A1 (D.B.), 1F32HL170591-01 (L.J.B.), the Howard Hughes Medical Institute (Y.E.C., C.O.B., Hanna H. Gray Fellowship), the Leona M. and Harry B. Helmsley Charitable Trust (M.A.F.), the Chan Zuckerberg Biohub (C.O.B., M.A.F.), the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID) (Y.B.), Intramural Research Programs of the National Human Genome Research Institute and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (J.A.S. and H.H.K.), the Office of Research Infrastructure Program, Office of The Director, NIH under Award Number P51OD011107 (CNPRC, UC-Davis), the Department of Defense NDSEG Fellowship (P.V.L.), the Knight-Hennessy Fellowship (P.V.L.), the Fannie and John Hertz Foundation (D.B.L.) and fellowships from The Helen Hay Whitney Foundation (K.D.B. and M.I.M.).
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D.B. and M.A.F. conceived of the study with help from K.D.B., Y.E.C., Y.B. and C.O.B. D.B. performed most experiments, analysed the data, cloned and designed most constructs, developed the methodology, isolated strains and characterized their B cell responses, performed the flow cytometry analysis and all tetanus challenges. K.D.B., Y.E.C., S.C., I.G., J.A.S., Y.B., C.O.B. and M.A.F. helped with the methodology. K.D.B., Y.E.C., V.K.Y., P.V.L., A.J., E.T., T.T.D.N., J.M.S., A.D., A.Z. and L.J.B. assisted with the in vivo work. K.D.B., Y.E.C., V.K.Y., P.V.L., A.J., E.T., M.I.M., A.N., J.L.P., T.T.D.N., J.M.S., Y.E.L. and C.O.B. took part in the in vitro work. K.D.B., Y.E.C., V.K.Y., and A.V. aided the cloning and strain generation efforts. S.C., S.J., X.M. and D.B.L. performed the bioinformatic analyses with support from H.H.K., J.A.S., M.A.F. and D.B. T.P.T.P. isolated bacterial strains from NHPs with help from K.K.A.V.R., D.B. and A.N. The figures were generated by D.B., M.A.F., D.B.L. and S.C. Funding was acquired by D.B., K.D.B., Y.E.C., P.V.L., M.I.M., L.J.B., K.K.A.V.R., Y.B., C.O.B. and M.A.F. The study was supervised by D.B., K.D.B., Y.E.C., K.K.A.V.R., H.H.K., J.A.S., Y.B., C.O.B. and M.A.F. The original manuscript was drafted by D.B. and M.A.F and reviewed and edited by M.A.F., D.B., K.D.B., Y.E.C., P.V.L., E.T., T.T.D.N., J.M.S., M.I.M., S.C., J.A.S., K.K.A.V.R. and C.O.B.
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M.A.F. is a cofounder of Kelonia and Revolution Medicines, a member of the scientific advisory boards of the Chan Zuckerberg Initiative, NGM Biopharmaceuticals and TCG Laboratories/Soleil Laboratories, and an innovation partner at The Column Group. D.B., Y.E.C., K.D.B., P.V.L., M.I.M., C.O.B., Y.B. and M.A.F. are inventors on patent applications submitted by Stanford University and the Chan Zuckerberg Biohub that cover methods for using engineered bacteria to elicit antigen-specific immune cells.
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Extended data figures and tables
Extended Data Fig. 1 Colonization with S. epidermidis induces a systemic B cell response.
a, Colony-forming units (CFUs) per ear for the experiment shown in Fig. 1a. Two independent experiments, all mice shown. b, S. epidermidis LM088 was stained with serum from mice that were colonized (blue) or not (naive, grey) with the same strain for 6 weeks and analysed by flow cytometry. Representative of three independent experiments. Bacteria were gated on Syto9 positive cells. c, CFUs for the experiment shown in Fig. 1b. Two independent experiments, all mice shown. d, CFUs for experiment shown in Fig. 1g. Two independent experiments, all mice shown. e, Gating strategy for germinal centre B cells as shown in Fig. 1e.
Extended Data Fig. 2 Antibody response against the native microbiome.
a, Representative example of a dot blot analysis using serum from mice housed at Charles River (CR) room H44, Taconic CC facility or at Jackson against a panel of strains isolated from the same mice. b, Bacterial ELISA of Staphylococcus xylosus and Streptococcus sp. isolated from Jackson and Taconic (Tac) GT mice respectively against a panel of serum from CR, Tac and Jackson. N = 5-15, one to three independent cages pooled. c, SPF mice (Taconic SD) were colonized for five weeks with murine strains of S. xylosus, Staphylococcus nepalensis or Staphyloccocus/Mammaliicoccus lentus and serum antibody titres were measured by ELISA. Colonization efficiency was confirmed at the experimental endpoint by CFUs (data not shown). d, Same as b but against two isolates of S./M. lentus isolated from Tac GT or CR H44. e, ELISA using serum from 11 healthy donors against a panel of commensals and environmental strains. f, Same as e but using the serum of 10 healthy non-human primates (NHP). e,f, One representative of three independent experiments. e,f, P values were calculated using one-way ANOVA with Tukey correction for multiple comparisons. Uncropped dot blot images can be found in Supplementary Fig. 2a.
Extended Data Fig. 3 Identification of the minimal epitope in the B ___domain of Aap.
a, Schematic of LM088 Aap protein truncations for expression in E. coli. b, Immunoblot analysis of the constructs shown in a using anti-HA antibody to assess expression (induced by IPTG), using serum from naive mice or mice colonized with LM088. (1 = amino acid repeat ___domain, 2 = parallel β-helix ___domain, and 3 = one repeat of the B ___domain). c, Schematic of the B ___domain truncations fused to superfolder GFP (sGFP) for expression in E. coli. d, Immunoblot analysis of the truncations shown in c using anti-HA antibody or the serum of mice colonized with LM088. e, Minimal epitope identified in the B ___domain and the peptides synthesized and tested in f. f, Dot blot analysis of the biotinylated peptides shown in e using streptavidin-HRP or the serum of mice colonized with LM088. All blots are representative of three independent experiments. g, Immunoblot of cell lysate (L) and culture supernatant (S) of LM088 and LM088 Δaap using serum from mice colonized with LM088 or LM088 Δaap (from the mice shown in Fig. 2j). Representative of two independent experiments. Uncropped immunoblot and dot blot images can be found in Supplementary Figs. 2b–d and 3a,b.
Extended Data Fig. 4 Redirecting the B cell response to S. epidermidis using engineered strains.
a, 6–10-week-old SPF mice were colonized for 6 weeks with S. epidermidis strain recombinantly expressing different tetanus toxin fragment C (TTFC) fusion proteins. The constructs were built in either WT LM087 (black) or LM088 Δaap (purple) background strain. b, Serum titres against TTFC six weeks post colonization, and c, TTFC specific IgA in nasal washes (undiluted washes). N = 8/group, two independent experiments pooled. Of note, for clarity, panel b shows medians and panel c displays means. The dashed lines show the corresponding median (b) and mean (c) values for strain Δaap + Aap-TTFC (wTTFC) (see Fig. 3d, which was run together with the data shown in Extended Data Fig. 4). d,e, Colony-forming units (CFUs) at the experimental endpoint for the constructs described in a on BHI (d) or BHI chlor (e). p = peptidoglycan targeting (i.e. LysM-2x TTFC). LOD = limit of detection.
Extended Data Fig. 5 Neonatal precolonization does not prevent a subsequent response to the same strain engineered to express TTFC.
a, Neonate SPF mice were colonized, or not, with the background strain Δaap every other day starting at day 7 for 1 week and subsequently once per week until day 42. The mice were rested for 2 weeks and subsequently colonized with wTTFC as adults following the typical schedule (every other day for 1 week following by one boost per week for a total of 6 weeks). At the experimental endpoint (day 98), serum was harvested and tested for the presence of TTFC-specific antibodies by ELISA (b). c, Colony-forming units at the end point on BHI (left) and BHI chlor (right). Of note, from day 7–21 the whole body of neonates and their mothers was colonized. After day 21, only the head was colonized. P values were calculated by unpaired two-sided Student t tests.
Extended Data Fig. 6 The SpyCatcher system can be used to conjugate proteins to the surface of S. epidermidis.
a, Coomassie staining of purified superfolder GFP (sGFP)-SpyTag003. b, S. epidermidis LM087 was engineered to express either a fusion between Aap and SpyCatcher (Aap-sc, yellow) or a catalytically dead version of SpyCatcher (Aap-sc*, grey). Both strains were incubated with 0.1, 0.3 or 1 mg ml−1 of sGFP-SpyTag for 2, 5, 15 or 60 min and visualized by flow cytometry. c, Surface expression of the transgene Aap-sc or Aap-sc* using a HA tag present on the construct for two different colonies (col) each. d, Coomassie staining of purified tetanus toxin fragment C (TTFC)-SpyTag003. e,f, Surface expression of Aap-sc and Aap-TTFC transgene on the corresponding strains as visualized on a flow plot (e) or the mean fluorescence intensity for four different colonies (f). a,d, One representative gel of at least three independent experiments. Uncropped Coomassie gels can be found in Supplementary Fig. 3c,d.
Extended Data Fig. 7 Quantification of the conjugation efficiency of TTFC to the Aap-sc strain.
a, Gating strategy for bacterial flow cytometry analysis. b, Schematic of Aap-sc* and conjugated Aap-sc-TTFC used in this figure. Both strains contain a HA tag fused to the SpyCatcher ___domain and TTFC-SpyTag003 a FLAG tag. c, Show the conjugation efficiency to TTFC-SpyTag003 for Aap-sc and Aap-sc* as measured by flow cytometry. d, Representative quantification experiment: (left) quantification beads and (right) four different colonies of Aap-sc conjugated to TTFC-SpyTag003. e, Quantification of the number of TTFC-SpyTag003 conjugated per bacteria and total amount of antigen given to mice per colonization for 6 colonies pooled from two different experiments. f, Formula used to calculate the total quantity of TTFC given per colonization using the #TTFC/bacterium calculated in d, e.
Extended Data Fig. 8 Aap-sc-TTFC elicit potent antibody response against TTFC systemically and at mucosal surface.
a, Experimental design. To ensure that conjugation of TTFC to the bacteria (i.e. Aap-sc) was necessary to induce antibodies against TTFC, we tested whether application of TTFC topically alone was sufficient to induce a response against TTFC. To identify the right amount of TTFC to apply, we first quantified the total amount of antigen given to mice when provided as Aap-sc-TTFC (see Extended Data Fig. 7). We estimated that each Aap-sc bacterium was conjugated to 30–60,000 TTFC molecules, corresponding to ~1 μg of TTFC per mouse per colonization. For topical administration, we thus used 5 μg of TTFC per application to be conservative. b, From top to bottom: TTFC-specific IgG titres in the serum, IgG in bronchoalveolar lavage (BAL) fluid, IgA in BAL fluid, and IgA in nasal washes for 13 (left), 3 (middle) or 2 (right) colonizations. c, Colony-forming units (CFUs) for the mice shown above in b and in Fig. 4f. All mice shown from two independent experiments.
Extended Data Fig. 9 Serum from Aap-sc-TTFC-colonized mice is protective against tetanus toxin challenge but not pure TTFC alone.
a, Experimental design. Mice received 5 μg of topical TTFC per application 13, 3 or 2 times, or were injected with mi3-TTFC intramuscularly twice. After 6 weeks, all mice received a lethal dose of tetanus toxin (150 ng/kg). b, Survival after challenge for the experiment described in a. c, Experimental design. Mice were injected with a lethal dose of tetanus toxin (110 ng/kg) preincubated with serum from mice colonized with either Aap-sc-TTFC or Aap-sc for 6 weeks. d, Survival curves for the experiment described in c. P values were calculated with log-rank tests. All mice shown for two independent experiments.
Extended Data Fig. 10 A reduced number of colonizations with Aap-sc-TTFC still elicits protective responses against tetanus toxin.
a, Experimental design. SPF mice were colonized with either Aap-sc or Aap-sc-TTFC 13 times (bacterial culture at OD600 = 6) on the head or 2, 5, or 6 times (bacterial culture at OD600 = 12) on the whole body. b, Antibody titres against TTFC in the nasal washes and bronchoalveolar lavage (BAL) fluid. c, Colony-forming units (CFUs) for mice shown in b and in Fig. 4i. d, Experimental design for the challenge experiment shown in e. e, Survival curves after receiving 1000 ng/kg of tetanus toxin. Of note, ~170 h post injection, 1/8 mice in the wTTFC group (5 and 6 colonizations) developed mild tetanus symptoms which never reached the humane endpoint (see *). P values were calculated log-ranked tests. All mice shown from two independent experiments.
Supplementary information
Supplementary Information
Supplementary Figs. 1–8.
Supplementary Table 1
Further information about reagents. Information about the bacterial strains, plasmids, sequences and antibodies used in this study.
Supplementary Data
Source Data for Supplementary Fig. 4.
Supplementary Data
Source Data for Supplementary Fig. 8.
Supplementary Video 1
Mice colonized with wDT are not protected against a lethal dose of tetanus toxin. Mice were colonized with S. epidermidis ∆aap expressing wDT (a catalytic mutant of diphtheria toxin) for 6 weeks and challenged with a lethal dose of tetanus toxin (150 ng kg−1) (mice shown in Fig. 3f). The video was recorded 44 h postinjection of the toxin and is representative of two independent experiments.
Supplementary Video 2
Mice colonized with wTTFC are protected against a lethal dose of tetanus toxin. Mice were colonized with S. epidermidis ∆aap expressing wTTFC for 6 weeks and challenged with a lethal dose of tetanus toxin (150 ng kg−1) (mice shown in Fig. 3f). The video was recorded 44 h postinjection (same experiment and time as mice shown in Supplementary Video 1) of the toxin and is representative of two independent experiments.
Supplementary Video 3
Mice colonized with wTTFC do not develop pathology after injection of a lethal dose of tetanus toxin. Mice were colonized with S. epidermidis ∆aap expressing wTTFC for 6 weeks and challenged with a lethal dose of tetanus toxin (150 ng kg−1) (mice shown in Fig. 3f). The video was recorded more than 200 h postinjection (same mice as shown in Supplementary Video 2) and is representative of two independent experiments.
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Bousbaine, D., Bauman, K.D., Chen, Y.E. et al. Discovery and engineering of the antibody response to a prominent skin commensal. Nature 638, 1054–1064 (2025). https://doi.org/10.1038/s41586-024-08489-4
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DOI: https://doi.org/10.1038/s41586-024-08489-4
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