Engineered immunogen binding to alum adjuvant enhances humoral immunity

Abstract

Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Phosphoserine affinity tags enable tunable binding of immunogens to aluminum hydroxide adjuvant.
Fig. 2: Alum-binding pSer antigens elicit enhanced humoral responses in vivo.
Fig. 3: Antigen-specific B cells efficiently take up pSer-antigen bound to alum particles in vivo.
Fig. 4: B cell activation by alum nanoparticles.
Fig. 5: pSer-antigen:alum nanoparticles recruit rare B cells to GCs and induce somatic hypermutation and memory.
Fig. 6: Enhanced humoral responses to HIV Env trimer immunogens elicited by pSer-antigen:alum immunization.

Data availability

All requests for raw and analyzed data and materials are promptly reviewed by the MIT Technology Licensing Office to verify whether the request is subject to any intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a Material Transfer Agreement.

References

  1. 1.

    Rappuoli, R. & Aderem, A. A 2020 vision for vaccines against HIV, tuberculosis and malaria. Nature 473, 463–469 (2011).

  2. 2.

    Plotkin, S. A. Correlates of protection induced by vaccination. Clin. Vaccine Immunol. 17, 1055–1065 (2010).

  3. 3.

    Reed, S. G., Orr, M. T. & Fox, C. B. Key roles of adjuvants in modern vaccines. Nat. Med. 19, 1597–1608 (2013).

  4. 4.

    Moyer, T. J., Zmolek, A. C. & Irvine, D. J. Beyond antigens and adjuvants: formulating future vaccines. J. Clin. Invest. 126, 799–808 (2016).

  5. 5.

    Kool, M., Fierens, K. & Lambrecht, B. N. Alum adjuvant: some of the tricks of the oldest adjuvant. J. Med. Microbiol. 61, 927–934 (2012).

  6. 6.

    McKee, A. S. & Marrack, P. Old and new adjuvants. Curr. Opin. Immunol. 47, 44–51 (2017).

  7. 7.

    HogenEsch, H., O’Hagan, D. T. & Fox, C. B. Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want. NPJ Vaccines 3, 51 (2018).

  8. 8.

    Francica, J. R. et al. Analysis of immunoglobulin transcripts and hypermutation following SHIV(AD8) infection and protein-plus-adjuvant immunization. Nat. Commun. 6, 6565 (2015).

  9. 9.

    Khurana, S. et al. Vaccines with MF59 adjuvant expand the antibody repertoire to target protective sites of pandemic avian H5N1 influenza virus. Sci. Transl. Med. 2, 15ra15 (2010).

  10. 10.

    Leroux-Roels, G. et al. Impact of adjuvants on CD4(+) T cell and B cell responses to a protein antigen vaccine: results from a phase II, randomized, multicenter trial. Clin. Immunol. 169, 16–27 (2016).

  11. 11.

    Hansen, B., Sokolovska, A., HogenEsch, H. & Hem, S. L. Relationship between the strength of antigen adsorption to an aluminum-containing adjuvant and the immune response. Vaccine 25, 6618–6624 (2007).

  12. 12.

    Morefield, G. L. et al. Effect of phosphorylation of ovalbumin on adsorption by aluminum-containing adjuvants and elution upon exposure to interstitial fluid. Vaccine 23, 1502–1506 (2005).

  13. 13.

    Iyer, S., HogenEsch, H. & Hem, S. L. Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production. Vaccine 21, 1219–1223 (2003).

  14. 14.

    HogenEsch, H. Mechanisms of stimulation of the immune response by aluminum adjuvants. Vaccine 20 (Suppl. 3), S34–S39 (2002).

  15. 15.

    Weissburg, R. P. et al. Characterization of the Mn Gp120 Hiv-1 vaccine: antigen-binding to alum. Pharmaceut. Res. 12, 1439–1446 (1995).

  16. 16.

    Noe, S. M., Green, M. A., HogenEsch, H. & Hem, S. L. Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response. Vaccine 28, 3588–3594 (2010).

  17. 17.

    Gupta, R. K., Chang, A. C., Griffin, P., Rivera, R. & Siber, G. R. In vivo distribution of radioactivity in mice after injection of biodegradable polymer microspheres containing 14C-labeled tetanus toxoid. Vaccine 14, 1412–1416 (1996).

  18. 18.

    Sok, D. et al. Priming HIV-1 broadly neutralizing antibody precursors in human Ig loci transgenic mice. Science 353, 1557–1560 (2016).

  19. 19.

    Jardine, J. G. et al. HIV-1 vaccines. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science 349, 156–161 (2015).

  20. 20.

    Jardine, J. et al. Rational HIV immunogen design to target specific germline B cell receptors. Science 340, 711–716 (2013).

  21. 21.

    Jardine, J. G. et al. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen. Science 351, 1458–1463 (2016).

  22. 22.

    Flarend, R. E. et al. In vivo absorption of aluminium-containing vaccine adjuvants using 26Al. Vaccine 15, 1314–1318 (1997).

  23. 23.

    Harris, J. R. et al. Alhydrogel(R) adjuvant, ultrasonic dispersion and protein binding: a TEM and analytical study. Micron 43, 192–200 (2012).

  24. 24.

    Abbott, R. K. et al. Precursor frequency and affinity determine B cell competitive fitness in germinal centers, tested with germline-targeting HIV vaccine immunogens. Immunity 48, 133–146 (2018).

  25. 25.

    Havenar-Daughton, C. et al. The human naive B cell repertoire contains distinct subclasses for a germline-targeting HIV-1 vaccine immunogen. Sci. Transl. Med. 10, eaat0381 (2018).

  26. 26.

    Tze, L. E. et al. CD83 increases MHC II and CD86 on dendritic cells by opposing IL-10–driven MARCH1-mediated ubiquitination and degradation. J. Exp. Med. 208, 149–165 (2011).

  27. 27.

    Kulp, D. W. et al. Structure-based design of native-like HIV-1 envelope trimers to silence non-neutralizing epitopes and eliminate CD4 binding. Nat. Commun. 8, 1655 (2017).

  28. 28.

    Steichen, JonM. et al. HIV vaccine design to target germline precursors of glycan-dependent broadly neutralizing antibodies. Immunity 45, 483–496 (2016).

  29. 29.

    Havenar-Daughton, C., Lee, J. H. & Crotty, S. Tfh cells and HIV bnAbs, an immunodominance model of the HIV neutralizing antibody generation problem. Immunol. Rev. 275, 49–61 (2017).

  30. 30.

    Pauthner, M. et al. Elicitation of robust tier 2 neutralizing antibody responses in nonhuman primates by HIV envelope trimer immunization using optimized approaches. Immunity 46, 1073–1088 (2017).

  31. 31.

    Drane, D., Gittleson, C., Boyle, J. & Maraskovsky, E. ISCOMATRIX adjuvant for prophylactic and therapeutic vaccines. Expert Rev. Vaccines 6, 761–772 (2007).

  32. 32.

    Bianchi, M. et al. Electron-microscopy-based epitope mapping defines specificities of polyclonal antibodies elicited during HIV-1 BG505 envelope trimer immunization. Immunity 49, 288–300 (2018).

  33. 33.

    Egan, P. M., Belfast, M. T., Gimenez, J. A., Sitrin, R. D. & Mancinelli, R. J. Relationship between tightness of binding and immunogenicity in an aluminum-containing adjuvant-adsorbed hepatitis B vaccine. Vaccine 27, 3175–3180 (2009).

  34. 34.

    Lu, F., Boutselis, I., Borch, R. F. & Hogenesch, H. Control of antigen-binding to aluminum adjuvants and the immune response with a novel phosphonate linker. Vaccine 31, 4362–4367 (2013).

  35. 35.

    Kool, M. et al. Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. J. Immunol. 181, 3755–3759 (2008).

  36. 36.

    Li, H., Willingham, S., Ting, J. & Re, F. Cutting edge: inflammasome activation by alum and alum’s adjuvant effect are mediated by NLRP3. J. Immunol. 181, 17 (2008).

  37. 37.

    Hornung, V. et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9, 847–856 (2008).

  38. 38.

    Eisenbarth, S. C., Colegio, O. R., O’connor, W., Sutterwala, F. S. & Flavell, R. A. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453, 1122–1126 (2008).

  39. 39.

    Ramanathan, V. D., Badenoch-Jones, P. & Turk, J. L. Complement activation by aluminium and zirconium compounds. Immunology 37, 881–888 (1979).

  40. 40.

    Guven, E., Duus, K., Laursen, I., Hojrup, P. & Houen, G. Aluminum hydroxide adjuvant differentially activates the three complement pathways with major involvement of the alternative pathway. PLoS ONE 8, e74445 (2013).

  41. 41.

    Flach, T. L. et al. Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity. Nat. Med. 17, 479–487 (2011).

  42. 42.

    Ghimire, T. R., Benson, R. A., Garside, P. & Brewer, J. M. Alum increases antigen uptake, reduces antigen degradation and sustains antigen presentation by DCs in vitro. Immunol. Lett. 147, 55–62 (2012).

  43. 43.

    Hu, J. K et al. Murine antibody responses to cleaved soluble HIV-1 envelope trimers are highly restricted in specificity. J Virol. 89, 10383–10398 (2015).

  44. 44.

    Attard, T. J., O’Brien-Simpson, N. M. & Reynolds, E. C. Identification and suppression of β-elimination byproducts arising from the use of Fmoc-Ser(PO3Bzl,H)-OH in peptide synthesis. Int. J. Pept. Res. Ther. 15, 69–79 (2009).

  45. 45.

    Weaver, G. C. et al. In vitro reconstitution of B cell receptor–antigen interactions to evaluate potential vaccine candidates. Nat. Protoc. 11, 193 (2016).

  46. 46.

    Lövgren-Bengtsson, K. & Morein, B. in Methods in Molecular Medicine, Vaccine Adjuvants: Preparation Methods and Research Protocols, Vol. 42 (ed. O’Hagan, D.) 239–258 (Humana Press, 2000).

  47. 47.

    Wei, X. et al. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother. 46, 1896–1905 (2002).

  48. 48.

    Wei, X. et al. Antibody neutralization and escape by HIV-1. Nature 422, 307–312 (2003).

  49. 49.

    Sarzotti-Kelsoe, M. et al. Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J. Immunol. Methods 409, 131–146 (2014).

  50. 50.

    Platt, E. J., Wehrly, K., Kuhmann, S. E., Chesebro, B. & Kabat, D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J. Virol. 72, 2855–2864 (1998).

  51. 51.

    Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171 (2014).

  52. 52.

    Rosales, S. L. et al. in Type 2 Immunity (ed. Reinhardt, R. L.) 275–301 (Springer, 2018).

  53. 53.

    Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

  54. 54.

    Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411 (2018).

  55. 55.

    Finak, G. et al. MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 16, 278 (2015).

  56. 56.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

  57. 57.

    Liberzon, A. et al. The molecular signatures database Hallmark gene set collection. Cell Syst. 1, 417–425 (2015).

  58. 58.

    Yu, D., Cozma, D., Park, A. & Thomas-Tikhonenko, A. Functional Validation of genes implicated in lymphomagenesis: an in vivo selection assay using a Myc-induced B cell tumor. Ann. NY Acad. Sci. 1059, 145–159 (2005).

  59. 59.

    Zhu, X. et al. Analysis of the major patterns of B cell gene expression changes in response to short-term stimulation with 33 single ligands. J. Immunol. 173, 7141–7149 (2004).

  60. 60.

    Lee, J. A. et al. Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation. BMC Bioinformatics 7, 237 (2006).

  61. 61.

    Busse, C. E., Czogiel, I., Braun, P., Arndt, P. F. & Wardemann, H. Single-cell based high-throughput sequencing of full-length immunoglobulin heavy and light chain genes. Eur. J. Immunol. 44, 597–603 (2014).

Download references

Acknowledgements

This work was supported in part by the NIAID under awards UM1AI100663 and UM1AI144462 (to D.J.I., W.R.S., S.C. and D.R.B.), AI125068 (to S.C. and D.J.I.), AI048240 (to D.J.I.), K99AI145762 (to R.K.A.) and AI113867 (to W.R.S.), the Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute, the Ragon Institute of MGH, MIT and Harvard and by the International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Consortium (NAC) and Center (to D.R.B., A.B.W. and W.R.S.); and through the Collaboration for AIDS Vaccine Discovery funding for the IAVI NAC Center (to D.R.B., A.B.W. and W.R.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.J.I. is an investigator of the Howard Hughes Medical Institute.

Author information

T.J.M., D.J.I., Y.K. and S.C. planned and designed experiments and analyzed data. W.R.S., D.W.K. and S.M. designed and produced immunogens. T.J.M., W.A., J.Y.H.C., N.W., H.A.S., S.H.-D.L.M., S.L., G.S., R.K.A., M.B.M., N.L. and Y.A. performed experiments. J.N.B. and D.R.B. carried out neutralization analysis of rabbit sera. H.L.T. and A.B.W. performed cryoTEM analysis of rabbit sera. T.J.M., D.J.I., W.R.S., Y.K. and S.C. wrote the manuscript. T.J.M., D.J.I., Y.K. and S.C. conceptualized the project. D.J.I. and S.C. provided research supervision.

Correspondence to Darrell J. Irvine.

Ethics declarations

Competing interests

D.J.I., T.J.M., Y.K., S.C. and W.R.S. are named as inventors on patent applications filed by MIT and The Scripps Research Institute related to the data presented in this work.

Additional information

Peer review information Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Extended data

Extended Data Fig. 1 Phosphoserines mediate protein binding to alum and influence immune response.

(a) Unmodified cytochrome-C protein or an equivalent concentration of cytochrome-C conjugated with a single pSer linker containing 1, 2, or 4 phosphoserine groups was mixed with 100 µg/mL Alhydrogel in 10 mM MOPS buffer. Alum was separated by centrifugation and bound protein was detected using absorbance at 410 nm compared to a standard curve of known protein concentration. (b) Peptide linkers of equivalent molecular size were prepared, composed of an azide functional group linked to either 4 phosphoserines, or 3 serines and 1 phosphoserine. These linkers were coupled to DBCO-modified phycoerythrin (PE) via copper-free click chemistry at a range of total linkers per protein. (c) pSer/Ser-conjugated PE (5 µg/mL) with varying numbers of linkers per protein was mixed with alhydrogel (50 µg/mL) for 30 min, followed by addition of mouse serum (final concentration 10% vol/vol) for 24 hr. Protein bound to alum after this incubation was assessed by fluorescence spectroscopy. (n=3 samples/group). Data represents mean ± SD. (d) PAGE gel of eOD protein reacted with pSer4-eOD. PNGase treatment was used with eOD to remove glycans prior to running gel. Representative gel from two experiments. Gel image is cropped. (e) eOD proteins (10 µg/mL) conjugated with a single 4-residue serine linker, or pSer linkers containing 1-8 phosphoserines, were incubated with AlHydrogel (100 µg/mL) for 30 min in TBS at 25 °C, followed by measurement of bound protein by fluorescence. (n=3 samples/group). Center lines represent mean and error bars represent SD. (f) Day 63 sera from BALB/c mice (n=5 animals/group) immunized with eOD (black) or pSer4-eOD (red) were tested for binding to pSer tags. Plates were coated with pSer4-cytochrome c to quantify the pSer-specific response. Data represents mean ± SD. (g, h) Germinal center B cells isolated from the inquinal lymph nodes of BALB/c mice (n= 10 animals/group, pooled from 2 experiments) 9 days after immunization with eOD variants and Al-Hydrogel. Cell counts are provided for GC B cells (g) and eOD-specific GC B cells (h). Center lines represent mean and error bars represent SD. Statistical tests were performed using one-way ANOVA with Tukey’s post test.

Extended Data Fig. 2 Antigen-specific B cells internalize pSer-antigens bound to alum particles in vitro.

(a) Schematic of potential release of free antigen vs. release of antigen-decorated alum particles at the injection site. (b) glVRC01-expressing human B cells were mixed with eOD (50 nM) and alum (10 µg/mL), or alum alone (10 µg/mL). Shown is calcium signaling in B cells following addition of antigen/alum at 60 sec (arrowhead) by the Fluo-4 fluorescence reporter. (c, d) Alum was labeled by mixing with Cy3-pSer4. glVRC01-expressing B cells were then incubated with fluorescent eOD (50 nM) and fluorophore-tagged alum (10 µg/mL) for 1 hour, and then imaged by confocal microscopy. (scale bars = 10 µm). Experiment was performed in two times, showing representative images from one experiment.

Extended Data Fig. 3 Alum particles are internalized by B cells in vitro.

(a) TEM images of sections of Ramos B cells in the absence of alum. Representative images are shown from a total of 20 cells imaged. (b) glVRC01-expressing Ramos B cells (2 million/mL) were incubated with pSer8-eOD (1 ug/mL) and alum (10 ug/mL) for 1 hour prior to fixation. Arrowheads point to electron dense alum nanofiber clusters. Representative images are shown from a total of 25 cells imaged.

Extended Data Fig. 4 Alum particles traffic to lymph nodes and are internalized by antigen presenting cells in vivo.

(a) Structure of IR680-pSer4 conjugate, synthesized by Cu-free click chemistry to directly label alum. (b) pSer-dye labeling of alum is stable even following incubation in serum. IR680-pSer4 conjugate was incubated either alone or with alum for 30 minutes, and then 10% mouse serum was added, and the solution was incubated at 37 °C for 72 hours. Data represents the fluorescence measurements of the supernatant after centrifugation to remove any dye remaining bound to alum. Other dyes (Cy3-DBCO and AlexaFluor488-DBCO) were conjugated in the same manner. (n=3 samples/group) Center lines and error bars represent mean and standard deviation, respectively. (c, d) Groups of BALB/c mice (n=5/group) were immunized with 5 µg IR800 dye-labeled Ser4-eOD (c) or pSer8-eOD (d), each mixed with 50 µg IR680-labeled alum, and total fluorescence from dLNs was measured in excised tissues at serial time points. Center lines represent mean. (eh) BALB/c mice (n=5 mice per group for naïve and day 3 groups, n=4 mice per group for day 7 groups) were immunized with pSer8-eOD:alum or Ser4-eOD:alum by s.c. injection, and flow cytometry was performed on draining lymph nodes after 1 or 7 days. Alum was labeled with pSer4-AF488, and eOD was labeled with AF647. Center lines and error bars represent mean and standard deviation, respectively. (i) ICP-MS measurements of aluminum in inguinal LNs of naïve mice and mice that were immunized s.c. 3 or 8 days prior to measurement. (n=5 mice/group). Center lines and error bars represent mean and standard deviation, respectively.

Extended Data Fig. 5 VRC01gHL cells take up eOD bound to alum particles in vivo.

(a) Gating strategy used to identify GFP+ VRC01gHL B cells. In vivo-acquired eOD antigen had been labeled with AF647 prior to injection. B cells were additionally stained ex vivo with BV711-labeled eOD to further confirm the specificity of the donor cells. (b, c) C57BL/6 mice adoptively transferred with 1x106 GFP+ CTV+ VRC01gHL B cells were immunized by i.p. injection of 5 µg AF647-labelled Ser4-eOD-GT8 or pSer8-eOD-GT8 together with 1 mg alum. (b) Representative flow cytometry analysis of VRC01gHL B cells. (c) Quantitation of antigen uptake over time. Lines indicate the mean. Data are representative of two independent experiments, n = 3 mice for d1/2 Ser4-eOD-GT8, n = 4 mice for d3 Ser4-eOD-GT8 and d1-3 pSer8-eOD-GT8. Statistical analysis was performed using Two-tailed Student t-test. (df) C57BL/6 mice adoptively transferred with 1x106 CTV+ VRC01gHL B cells were immunized by i.p. injection of 5 µg AF647-labelled Ser4-eOD-GT8 or pSer8-eOD-GT8 together with 1 mg pSer-AF488 labelled alum. (d) Representative flow cytometry plot of VRC0gHL cells. (e) Quantification of eOD-GT8 uptake by VRC01gHL cells. (f) Quantification of alum uptake by VRC0gHL cells. Bars represent the mean. Data combined from two independent experiments, n = 4 mice for unimmunized, n = 8 mice for Ser4-eOD-GT8 + alum, n = 7 mice for pSer8-eOD-GT8 + alum. Statistical analysis was performed using One-way ANOVA with Tukey’s post-test.

Extended Data Fig. 6 TEM of sorted B cells after immunization with eOD-GT5 or eOD-GT8.

(a, b) Mice were adoptively transferred with VRC01gHL B cells then immunized with fluorescent pSer8-eOD-GT5 and alum i.p. Two days after immunization, endogenous (a) and eOD-GT5+ VRC01gHL B cells (b) were sorted by flow cytometry, fixed, and sectioned for TEM imaging. 145 (a) and 153 (b) cells were analyzed, and representative images are shown. (c, d) Identical experimental conditions were used, with pSer8-eOD-GT8 as the antigen, for TEM imaging of endogenous (c) and eOD-GT8+ VRC01gHL B cells (d). 126 cells were imaged for (d) and 45 cells were imaged for (c). (e) Quantification of images from (c) and (d). Arrowheads point to electron dense alum clusters.

Extended Data Fig. 7 RNA-seq analysis of VRC01-class bnAb precursors.

a) Bulk RNA-seq analysis showing the row-wise z score of differentially expressed genes in VRC01gHL B cells from vaccinated mice compared to naive cell (2-fold cutoff, adj p < 0.01). n = 3 mice for group 1-4, n = 4 mice for group 5. (b) Venn diagrams summarizing bulk RNA-seq analysis showing the number of differentially expressed genes in VRC01gHL cells from each group compared to naive cells (1.5-fold cut-off, adj p <0.05, DeSeq2 pairwise comparisons). (c) t-Distributed stochastic neighboring embedding (tSNE) two-dimensional plot of single-cell RNA-seq data shows different clustering patterns of VRC01gHL cells from different immunization groups. n = 42 cells (4 mice) for naïve, n = 55 cells (4 mice) for Ser4-eOD, n = 53 cells (4 mice) for pSer-eOD (Ag–), n = 58 cells for pSer-eOD (Ag+) (4 mice), n = 35 cells (2 mice) for 60mer. Pooled data from two adoptive transfer experiments. (d) Normalized expression of MHC II genes and March1 by VRC01gHL cells. Horizontal lines in violin plots indicate the first quartile, the median, and the third quartile. Each circle represents a cell. n = 37 cells (2 mice) for Ser-eOD + alum, n = 37 cells (2 mice) for pSer-eOD + alum (Ag–), n = 46 cells (2 mice) for pSer-eOD + alum (Ag+), n = 35 cells (2 mice) for eOD-60mer. (eh) C57BL/6 mice adoptively transferred with 1x106 CTV+ VRC01gHL B cells were immunized by i.p. injection of 10 µg AF647-labelled Ser4-eOD-GT5 or pSer8-eOD-GT5 together with 1 mg alum. (e) Flow cytometry analysis showing changes in MHC II and CD83 expression patterns on VRC01gHL B cells. Number indicates percentage of CD83+ MHC IIhi VRC01gHL B cells. Two independent experiments were performed with similar results. (f) Bar graphs show percentage of CD83+ MHC IIhi VRC01gHL B cells in (E). Bars represent the mean. Pooled data from two experiments. n=7 for group 1 and 2, n=6 for group 3 and 4, n=3 for group 5. (g) Flow cytometry analysis showing changes in CD86 expression patterns on VRC01gHL B cells. Two independent experiments were performed with similar results. (h) Bar graphs show gMFI of CD86 on VRC01gHL cells in (G) normalized to unprimed group. Bars represent the mean. Data combined from two independent experiments. ***, p = 0.002; ****, p < 0.0001 by One-way ANOVA with Tukey’s post-test. (i) Day 28 sera from C57BL/6 mice and NLRP3 KO mice (n=5 animals/group) immunized with pSer8-eOD (red, black) or Ser4-eOD (grey) was analyzed by ELISA for eOD-specific IgG antibody responses. Statistical comparison by One-way ANOVA with Tukey’s post-test. (j) Day 14 sera from BL/6 mice (red) and IL18 KO mice (black) (n=5 animals/group) immunized with pSer8-eOD was analyzed for eOD-specific IgG antibody responses. p=0.69 by two-tailed Student’s t-test.

Extended Data Fig. 8 Flow cytometry gating for VRC01gHL B cell populations.

(a) Gating strategy for VRC01gHL GC B cells. (b) Gating strategy for GC-derived VRC01gHL memory B cells.

Extended Data Fig. 9 pSer-MD39 binds to alum, enhancing humoral and neutralization responses.

(a) After incubation of MD39 trimer with alum for 30 minutes, 10% mouse serum was added, and solution was incubated for 24 hours. Binding of MD39 to alum was determined by the presence of MD39 in the supernatant after pelleting alum by centrifugation. MD39 concentration was measured by ELISA relative to a standard curve. (n=3 samples/group). Center lines and error bars represent mean and SD, respectively. (b) Representative images of fluorescently labeled MD39 or pSer4-MD39 retention at the injection site after immunization with alum in BALB/c mice (n = 4 animals/group). (c) Quantification of IVIS images from (b). Data represents mean ± SD. (d) ISCOM-like saponin adjuvant does not inhibit pSer-immunogen binding to alum. Fluorescently labeled pSer4-eOD (10 ug/mL) was combined with either alum or alum and saponin nanoparticles (labeled isco). After incubation for 24 hours in the presence of 10% mouse serum, the percentage of unbound eOD was measured by fluorescence. The fluorescence signal of pSer4-eOD in the absence of alum was normalized to 100% (n=3 samples/group). Center lines and error bars refer to mean and SD, respectively. (e) BALB/c mice (n=10/group, pooled from two immunizations) were immunized with 5 µg MD39 or pSer4-MD39 mixed with 50 µg alum and 5 µg saponin adjuvant. IgG titers from individual mice at week 6. . Statistical analysis by two-tailed Student’s t-test of the log-transformed data. Data are represented as mean ± SD of the log-transformed data. (f) Representative images of bone marrow ELISPOT plates for antibody-secreting cells at 3 months post immunization using the same conditions describe in (e). Experiments were performed twice, after two separate immunizations (n=5 mice/group for each experiment). (g) Quantification of mean bone marrow MD39 trimer-specific ELISPOT responses described in (f). Statistical analysis by two-tailed Student’s t-test (n=5 animals/group). Center lines and error bars refer to mean and SD, respectively. (h) Flow cytometry gating for identification of Env trimer-specific memory B cells. (i) Groups of BALB/c mice (n=5 mice/group for MD39 and pSer-MD39, n=2 mice/group for naive) were immunized were immunized with 5 µg MD39 or pSer8-MD39 mixed with 50 µg alum and 5 µg saponin adjuvant on day 0. Shown are frequencies of MD39+ memory B cells from spleens at week 25. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. Center lines represent mean. (j, k) BALB/c mice (n=5 mice/group) were immunized with 5 ug antigen, and titers were measured four weeks after s.c. immunization. (j) Ser4-eOD, alum-binding pSer8-eOD, and eOD 60mer were used as antigens in combination with alum. Statistical analysis was performed using one-way ANOVA with Tukey’s post-test (n=5 mice/group) of the log-transformed data. Center lines and error bars refer to mean and SD, respectively, of the log-transformed data. (k) MD39 control, pSer4-MD39, and MD39-ferritin were used as antigens in combination with alum. Statistical analysis was performed using one-way ANOVA with Tukey’s post-test (n=5 mice/group) of the log-transformed data. Center lines and error bars refer to mean and SD, respectively, of the log-transformed data. (l) BALB/c mice (n=10 mice/group) were immunized with 5 µg MD39 or pSer4-MD39 mixed with 50 µg alum on day 0 with ISCOM-like adjuvant. His tag-specific IgG titers at day 63. Statistical analysis by two-tailed Student’s t-test of the log-transformed data. Center lines and error bars refer to mean and SD, respectively, of the log-transformed data. (m) Rabbits were immunized with MD39:alum or pSer8-MD39:alum on weeks 0 and 8 (n=6 rabbits/group). Purified IgG antibodies from rabbit sera collected on week 10 was measured for neutralization against autologous tier 2 virus. Statistical analysis by Mann-Whitney test. Center lines and error bars refer to mean and SD, respectively.

Extended Data Fig. 10 EM averages and reconstructions.

(a) Representative subset of single particle nsEM 2D class averages (left) and corresponding reconstructed 3D maps (right). The 3D reconstructions are an average composite of most particles in each dataset and reveal the epitopes targeted in each serum sample as labeled. (b) Per animal epitope analysis showing a single segmented Fab per epitope as determined by further 3D classification of average composite reconstructions shown in Supplementary Figure 7. Colors represent the region of trimer where Fabs bind (blue - V5, pink - N355/alpha2 helix, purple - base, and yellow - N611). Three separate rabbit sera were analyzed for both MD39 and pSer8-MD39 immunizations.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Moyer, T.J., Kato, Y., Abraham, W. et al. Engineered immunogen binding to alum adjuvant enhances humoral immunity. Nat Med 26, 430–440 (2020). https://doi.org/10.1038/s41591-020-0753-3

Download citation