A modular vaccine platform for optimized lipid nanoparticle mRNA immunogenicity

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A modular vaccine platform for optimized lipid nanoparticle mRNA immunogenicity

Data availability

The primary data related to the results of this study can be found within the paper and its Supplementary Information. Source data are provided with this paper.

Code availability

The code used for protein sequence analysis in this study is available on GitHub (https://github.com/fangzhe3/MVP_code)52.

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Acknowledgements

This work was supported by discretionary funds and a Cancer Research Institute Lloyd J. Old STAR Award (CRI4964) to S.C. We acknowledge support from various Yale core facilities: the Systems Biology Institute, Department of Genetics, Department of Immunobiology, Yale School of Medicine Dean’s Office and Office of the Vice Provost for Research. We thank X. Chen, L. Chen, F. Fenteany, L. Lawres, L. Zhou, K. Tang, X. Zhou, P. Ren and many other laboratory members and/or colleagues for providing reagents, suggestions and technical assistance. Z. Fang is supported by the Canadian Institutes of Health Research (funding reference number 194053).

Author information

Author notes

  1. These authors contributed equally: Zhenhao Fang, Valter S. Monteiro.

  2. These authors jointly supervised this work: Carolina Lucas, Sidi Chen.

Authors and Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    Zhenhao Fang, Changin Oh, Kawthar Al Janabi, Luciano Romero, Nabihah Ahsan, Luojia Yang, Lei Peng, Daniel DiMaio & Sidi Chen

  2. Systems Biology Institute, Yale University, West Haven, CT, USA

    Zhenhao Fang, Kawthar Al Janabi, Luciano Romero, Nabihah Ahsan, Luojia Yang, Lei Peng & Sidi Chen

  3. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA

    Zhenhao Fang, Kawthar Al Janabi, Luciano Romero, Nabihah Ahsan, Luojia Yang, Lei Peng & Sidi Chen

  4. Immunobiology Program, Yale University, New Haven, CT, USA

    Valter S. Monteiro, Carolina Lucas & Sidi Chen

  5. Yale College, New Haven, CT, USA

    Kawthar Al Janabi, Luciano Romero, Nabihah Ahsan & Sidi Chen

  6. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA

    Luojia Yang & Sidi Chen

  7. Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, USA

    Daniel DiMaio

  8. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Daniel DiMaio

  9. Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Daniel DiMaio & Sidi Chen

  10. Center for Infection and Immunity, Yale University, New Haven, CT, USA

    Carolina Lucas

  11. Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA

    Carolina Lucas

  12. Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  13. Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

Authors

  1. Zhenhao Fang
  2. Valter S. Monteiro
  3. Changin Oh
  4. Kawthar Al Janabi
  5. Luciano Romero
  6. Nabihah Ahsan
  7. Luojia Yang
  8. Lei Peng
  9. Daniel DiMaio
  10. Carolina Lucas
  11. Sidi Chen

Contributions

S.C. conceived of the study and designed it with Z.F. Z.F. performed most of the experiments and analysed the data. V.S.M. performed the VACV challenge experiment. C.O. prepared the HPV-transduced cell lines. K.A.J., L.R. and N.A. assisted with the experiments related to molecular cloning, ELISA and flow cytometry. L.Y. and L.P. conducted the experiments involving lentivirus production and transduction. Z.F., D.D., C.L. and S.C. wrote the paper with input from all authors. C.L. and S.C. supervised the study.

Corresponding authors

Correspondence to Carolina Lucas or Sidi Chen.

Ethics declarations

Competing interests

S.C. is a (co)founder of EvolveImmune Therapeutics, Cellinfinity Bio, MagicTime Medicine and Chen Consulting, all unrelated to this study. A patent application related to this study has been filed by Yale University. The patent titled ‘Compositions and Methods for Enhancement of mRNA Vaccine Performance and Vaccination Against Mpox’ (PCT International Appl. No. PCT/US2023/081090) covers the related biotechnology. The other authors declare no competing interests.

Peer review

Peer review information

Nature Biomedical Engineering thanks Anna Blakney and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Flow cytometry gating strategy to select A35R-positive 293T cells which overexpressed A35R ectodomain recombined with various N-term type II TM/Cs and were surface stained with PE anti-A35R antibody.

The 293T cells were gated at low and high threshold to define PE and PE-high populations, respectively (n = 3). The A35R TM/C + A35R ectodomain served as the internal control. The A35R ectodomains with endomembrane protein TM/Cs, such as TMX4, SEC11A, PM121, served as TM/C negative controls. The TM/C modules that outperformed A35R TM/C (orange) are highlighted in red or purple (color based on their located quadrants in Fig. 2a). Schematic (top right) created with BioRender.com.

Extended Data Fig. 2 Representative flow plots showing the gating strategies to define PE or PE-high positive 293T cells that overexpressed A29 with or without either [Spike SP] + type I TM/Cs (bottom panel) or Type II TM/Cs (top panel) and were surface stained with PE anti-A29 antibody.

The A35R TM/C + A29 served as the internal control, while the A29 with endomembrane protein type II TM/Cs, including TMX4 and SEC11A, served as TM/C negative controls. The TM/C modules that outperformed A35R TM/C (orange) are highlighted in red or purple (color based on their type I or type II topology). Schematic (top right) created with BioRender.com.

Extended Data Fig. 3 ELISA titration curves over serial dilutions of plasma collected on different days from mice immunized with 1 µg [CD8 SP]-M1Re LNP-mRNAs with different TM/Cs.

a, ELISA titration curves on days 0, 14, 21 and 35 (n = 5). The immunization schedule is shown at the top of the graph. b, correlation of antibody titers against M1R with corresponding antigen’s expression on 293T cells. The antigen expression as CST quantified by normalized PE integral (positive rate x positive MFI ratio) is plotted against anti-M1R antibody AUC titer. all data points except for the secreted M1Re is analyzed in linear regression. c, TM/C modules modulated the antibody and T cell response to LNP-mRNA of chimeric [CD8a SP]-M1Re in mice. The anti-M1R antibody titer on day 35 is plotted against Th1 response (IFN- γ as an indicator). Data are presented as mean ± SEM. Schematic in a created with BioRender.com.

Extended Data Fig. 4 The CST scores were calculated from normalized antigen surface expression and were used to evaluate SP and TM/C modules’ strength to promote different Mpox antigens translocation to cell surface.

a,c, The fusion of flag-tagged (a) or untagged (c) [CD8a SP]-A29 with different type I TM/Cs enhanced its surface expression in 293T cells (n = 3). b,d, Various N-term signal peptides increased flag tagged A29-[HLA TM/C] (b) or E8L (d) surface expression levels (n = 3). e,f, The CST score was used to evaluate the signal strength of different SP (e) or TM/C (f) modules at promoting antigen expression on 293T cell surface. It is derived from normalized antigen expression, of which largest value in each dataset was normalized to 1. e, Various signal peptides (SP) increased cell-surface expression of flag-tagged Mpox antigens, including A29, M1Re and E8L (n = 3). f, Different type I TM/Cs improved cell-surface expression of flag-tagged or untagged A29 and M1Re in 293 T cells (n = 3). Data are presented as mean ± SEM.

Extended Data Fig. 5 T cells from mice vaccinated with MVP-modified mRNA antigens showed higher cytokine secretion after VZV or HPV antigen stimulation.

PBMCs were collected on day 28 from mice vaccinated different LNP mRNAs and stimulated with VZV gE antigen or HPV E6, E7 peptides for 18 and 30 hours. The secreted cytokines in media were measured by beads-based immunoassays (seen methods). a, Cytokine secretion of T cells stimulated with VZV gE antigen for 18 h (top) and 30 h (bottom). b, Cytokine secretion of T cells stimulated with HPV E6 and E7 scanning peptide pools for 18 h (top) and 30 h (bottom). All statistics were derived from Dunnett’s multiple comparison test between controls (VZV gE full length or HPV16 E6 + E7) and other treatment groups. PMA/Ionomycin group was excluded from comparisons. Sample number of vaccination groups (except for PMA/Ionomycin) is 5. Data are presented as mean ± SEM. Dunnett’s comparison test was used to determine statistical significance between WT antigens and modified antigen groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Mouse icons created with BioRender.com.

Extended Data Fig. 6 Compared to E6 + E7 vaccination control, T cells collected on day 35 from mice vaccinated with MVP-modified E6 and E7 mRNA antigens showed higher target cell killing activity (a), activation induced markers (b) and cytokine secretion (c) after 18-hour co-culture with B16F10 target cells transduced with HPV16 wild-type E6 + E7 and pulsed with E6, E7 peptides.

a, T cells from mice vaccinated with MVP-modified E6, E7 mRNA antigens led to higher cytolysis of B16F10 target cells (n = 5). b, Activation induced markers were surface stained in T cells from different LNP mRNA vaccination groups after co-culture with target cells (n = 5). c, Cytokine secretion of T cells co-cultured with target cells for 18 hours (n = 5). The secreted cytokines in media were measured by beads-based immunoassays (seen methods). Target cell alone showed baseline levels of cytokines in media. Additional cytokines, such as IL-2 and IL-12 were added to media to maintain effector function of T cells. All statistics were derived from Dunnett’s multiple comparison test between HPV16 E6 + E7 and other treatment groups. PMA/Ionomycin group was excluded from comparisons. Sample number of vaccination groups is 5, except for PMA/Ionomycin and target cell. Data are presented as mean ± SEM. Dunnett’s comparison test was used to determine statistical significance between WT antigens and modified antigen groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Panel a and insets in b and c created with BioRender.com.

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Fang, Z., Monteiro, V.S., Oh, C. et al. A modular vaccine platform for optimized lipid nanoparticle mRNA immunogenicity. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01478-6

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