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Floss-based vaccination targets the gingival sulcus for mucosal and systemic immunization

Nature Biomedical Engineering (2025)Cite this article

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Abstract

The oral cavity is an accessible site for vaccination, but its sublingual and buccal sites have limited vaccine uptake. Here we show that flat tape dental floss can deliver vaccines through the junctional epithelium of the gingival sulcus, exploiting its naturally leaky properties. Floss-based vaccination delivered protein, inactivated virus, peptide-presenting immunogenic nanoparticles and messenger RNA. In mice, gold nanoparticles functionalized with a peptide derived from the ectodomain of the transmembrane matrix 2 protein of human influenza virus stimulated local lymph nodes, increased CD4+T cells in lymph nodes, lungs and spleen, and boosted antibody-secreting cells in the bone marrow. Floss-based immunization induced strong and sustained immune activation across multiple organs, robust systemic and mucosal antibody responses, and durable protection against lethal influenza infection, independent of age, food and liquid consumption. Floss-based vaccination was superior to sublingual and comparable with intranasal vaccination. In human participants, fluorescent dye delivered via floss picks effectively reached gingival sulcus, supporting clinical feasibility. These findings establish floss-based vaccination as a simple, needle-free strategy that enhances vaccine delivery and immune activation compared with existing mucosal immunization methods.

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Fig. 1: Coated floss enables delivery across the JE.
Fig. 2: Characterization of immune response after floss-based vaccination using Ova.
Fig. 3: Characterization of the immune response after floss-based vaccination using Inac. virus.
Fig. 4: Protective efficacy of M2e-based broadly protective influenza vaccine delivered with coated floss and effect of eating and drinking on the antibody response.
Fig. 5: Comparison of floss-based vaccination to the IN route.
Fig. 6: Floss-based immunization induces acute and chronic immune activation in different immune organs.
Fig. 7: Floss-based vaccination induces strong antibody responses in an age-independent manner and floss coated with GFP–mRNA induces antibody response in serum.
Fig. 8: Use of floss pick in humans.

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Data availability

All data that support the claims of this study are available in this article or its Extended Data and Supplementary Information. Source data are provided with this paper. For additional information, please contact the corresponding author at hsgill2@ncsu.edu.

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Acknowledgements

We acknowledge the support provided by the National Institutes of Health. H.S.G. discloses support for the research described in this study from the National Institutes of Health (grant no. R01AI137846), the National Institutes of Health (grant no. R01DE033759) and the Whitacre Endowed Chair in Science and Engineering. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We also thank C. Roseburd Magezi Ndamira, a graduate student of Gill lab at TTU, for her help in capturing Supplementary Movie 1.

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, Texas Tech University, Lubbock, TX, USA

    Rohan S. J. Ingrole, Akhilesh Kumar Shakya, Gaurav Joshi, Chang Hyun Lee, Lazar D. Nesovic & Harvinder Singh Gill

  2. Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA

    Gaurav Joshi & Harvinder Singh Gill

  3. Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA

    Richard W. Compans

  4. Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC, USA

    Harvinder Singh Gill

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Contributions

Conceptualization: R.S.J.I., A.K.S. and H.S.G. Methodology: R.S.J.I., A.K.S. and H.S.G. Investigation: R.S.J.I., A.K.S. and H.S.G. Human studies: R.S.J.I. Data analysis: R.S.J.I. and H.S.G. Creation of figures: R.S.J.I. and H.S.G. Funding acquisition: H.S.G. Project administration: H.S.G. Supervision: H.S.G. Writing—original draft: R.S.J.I. and H.S.G. Writing—review and editing: R.S.J.I., R.W.C. and H.S.G. G.J. prepared the influenza virus stocks used for challenge and for Inac. virus study. C.H.L. helped with design of the primers used for qRT–PCR study and in analysis of the qRT–PCR data. L.D.N. helped with design and analysis of flow cytometry experiment.

Corresponding author

Correspondence to Harvinder Singh Gill.

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Competing interests

H.S.G., R.S.J.I. and A.K.S. are coinventors on a patent related to targeting the JE for vaccination and allergen immunotherapy. H.S.G. and R.S.J.I. are coinventors on a patent related to the pipette-based coating of floss. A startup company is pursuing this technology. This startup company has licensed the patent related to targeting the JE for vaccination and allergen immunotherapy. R.S.J.I. has equity in this company. R.S.J.I. has joined this company by the time the revised version of the paper was submitted. However, findings in this article were not influenced by the company. This potential competing interest has been disclosed and is being managed by TTU. No part of this study was paid for by the company nor was the company involved in any data interpretation or analysis. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Flossing procedure ex-vivo.

a, Brightfield stereoscopic image of the jaw tissue of naïve mice (top panel) and the jaw tissue after placing flat tape floss between the bottom two incisors (bottom two panels). The floss rested in the cavity without damaging the surrounding muscle tissue.

Extended Data Fig. 2 IVIS-based temporal tracking and quantification of material delivered to the JE.

Mice were treated with Alexa660-tagged Ova, either coated on floss or administered in a drop-wise manner. Untreated (Naïve) mice were used as the negative control. All mice were provided ad libitum access to food and water post-treatment. IVIS images of naïve mice (a). IVIS images showing retention of Alexa660-tagged Ova at the delivery site in mice treated in a drop-wise manner (b) or treated with coated floss (c).

Extended Data Fig. 3 Ex-vivo temporal tracking and quantification of material delivered to the JE.

Rhodamine-tagged Ova was delivered to the gingival sulcus either using a coated floss or drop placement. At different time points, tissue was excised, washed, and imaged using a stereomicroscope. a, Brightfield and fluorescence images for flossed mice. b, Brightfield and fluorescent images for mice receiving a liquid drop.

Extended Data Fig. 4 Recall antibody response post-challenge in mice vaccinated with inactivated virus coated on the floss.

Mice were immunized with either 10 µg or 25 µg of Inac. A/Puerto Rico/8/34 virus (PR8) influenza virus coated on floss. Mice either received two or three immunizations. UnVacc. mice were used as a negative control. On day 57, mice including the UnVacc. group were challenged with 3×LD50 of influenza A PR8 virus. On day 4 post-challenge, different mucosal secretions such as fecal pellets, vaginal wash, and saliva were collected from mice. Mice were then euthanized, and LL and NW were collected from euthanized mice. Anti-Inac. virus specific IgG and IgA in fecal pellets (a), vaginal wash (b), and saliva (c). Anti-Inac. virus specific IgG in LL (d), and NW (e) of immunized (floss) or UnVacc. mice. Data are presented as the arithmetic mean ± SD of samples from individual animals. N = 4 to 5 mice in each group. The Mann–Whitney Unpaired test (two-tailed) was used to calculate p values for differences between groups.

Source data

Extended Data Fig. 5 Recall antibody response post-challenge in mice vaccinated with M2e-AuNP + CpG.

Mice were vaccinated on day 0 and day 21 with M2e-AuNP+CpG (1X) vaccine formulation either coated on floss or administered IN. UnVacc. mice were used as negative controls. On day 43, mice were challenged with 3×LD50 of influenza A H1N1pdm09 virus. On day 4 post-challenge, different mucosal secretions, such as vaginal wash and saliva, were collected from the mice. Mice were then euthanized, and LL and NW were collected from euthanized mice. Anti-M2e IgG and IgA in vaginal wash (a), and saliva (b), LL (c), and NW (d) of immunized (floss/ IN) or UnVacc. mice. N = 3 to 5 mice in each group. Data are presented as the arithmetic mean ± SD of samples from individual animals. The Mann–Whitney Unpaired test (two-tailed) was used to calculate p values for differences between groups.

Source data

Supplementary information

Supplementary Information

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Reporting Summary

Supplementary Movie 1

Floss coated with rhodamine-tagged Ova for flossing mice incisors ex vivo.

Supplementary Movie 2

Demonstration of flossing mice incisors, ex vivo, with rhodamine-tagged Ova.

Supplementary Movie 3

Demonstration of flossing in human and deposition of coatings in gingival sulcus.

Source data

Source Data Figs. 1–8 and Extended Data Figs. 4 and 5

For Fig. 1: FIG_1, FIG_2, FIG_3, FIG_4, FIG_5, FIG_6, FIG_7 and FIG_8. For extended data: ED_FIG4, ED_FIG5, Supplementary Fig. 1, Supplementary Fig. 2, Supplementary Fig. 3, Supplementary Fig. 4 and Supplementary Fig. 5.

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Ingrole, R.S.J., Shakya, A.K., Joshi, G. et al. Floss-based vaccination targets the gingival sulcus for mucosal and systemic immunization. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01451-3

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